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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 10 October 2016. Experimental completion date: 03 February 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride
EC Number:
947-079-6
IUPAC Name:
Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride
Test material form:
other: paste
Specific details on test material used for the study:
Identification: Methoxycinnamidopropyl Behendimonium Chloride
Physical state/Appearance: Off-white paste
Batch: INV-1603010
Purity: 29.73%
Expiry Date: 01 February 2017
Storage Conditions: Room temperature in darkness

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range Finding test
A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitve test
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates (replicates R1 – R4) at 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples of the control and each loading rate WAF test group were taken and stored frozen for further analysis if necessary.
Only samples at the No Observed Effect Loading Rate and above were analyzed.

Test solutions

Vehicle:
no
Details on test solutions:
Range-finding Test
Nominal amounts of test item (5.0, 50 and 500 mg) were each separately dispensed onto a glass slide suspended within 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs showed no micro-dispersions; however the WAFs were observed to be cloudy dispersions, which indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length) and filter paper. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions.

Initial Test
WAFs were prepared by separately dispensing nominal amounts of test item onto a glass slide suspended within 2.5 liters of reconstituted water (ISO) prior to the start of stirring. WAFs were stirred for 23 hours followed by a 1-Hour settling period prior to removal of the WAFs by mid-depth siphoning and filtration through a glass wool plug and filter paper. Due to the potential presence of undissolved test item, immobilized daphnia were examined microscopically in order to determine whether the effects seen were physiological or toxicological.
WAFs at 3.2, 10 and 32 mg/L loading rate were prepared by separately dispensing nominal amounts of test item onto a glass slide suspended within 2.5 liters of reconstituted water (Elendt M7) prior to the start of stirring. WAFs were stirred for 23 hours followed by a 1-Hour settling period prior to removal of the WAFs by mid-depth siphoning and filtration through a glass wool plug and filter paper. The 3.2 and 10 mg/L loading rate WAFs were diluted to produce the 0.32 and 1.0 mg/L loading rates, respectively. Due to the potential presence of undissolved test item, immobilized daphnia were examined microscopically in order to determine whether the effects seen were physiological or toxicological.

Definitive Test
Nominal amounts of test item (25, 45, 80, 140 and 250 mg) were prepared by separately dispensing nominal amounts of test item onto a glass slide suspended within 2.5 liters of reconstituted water (Elendt M7) to give the 10, 18, 32, 56 and 100 mg/L loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column, and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length) and filter paper. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing, which was followed by a piece of filter paper, and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no test item to be present.

Validation of Mixing Period
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item present in a WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and filtration through glass wool and filter paper, and samples taken for analysis.
The results are summarized as follows:

Nominal Loading Rate (mg/L): 100
Measured Concentration (mg/L) (24 hours): 25.3
Measured Concentration (mg/L) (96 hours): 30.0

It is evident from this work that increasing the stirring period did not significantly increase the amount of dissolved test item in the WAF, and so preparation of the WAF was maintained at 24 hours.
Visual observations of the WAFs indicated that not all of the undissolved test item could be removed by filtration through a glass wool plug and two filter papers. Therefore, any immobilized daphnia were to be examined microscopically in order to determine whether observed effects were physiological rather than toxicological.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3
Test temperature:
Temperature was maintained at 21 ºC to 22 ºC
Water temperature was recorded daily throughout the test. Temperature was measured using a Hanna Instruments HI 93510 digital thermometer
pH:
7.8 - 7.9
pH were recorded at the start and termination of the test. The pH were measured using a Hach Flexi handheld meter. There were no treatment related differences for pH.
Dissolved oxygen:
8.2 - 8.8 mgO2/L
Dissolved oxygen concentrations were recorded at the start and termination of the test. Dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the. There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
Range Finding Test: 1.0, 10 and 100 mg/L loading rates
Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of 0.676 and 4.37 mg/L, respectively. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions.

Initial Experiments: 10, 18, 32, 56 and 100 mg/L loading rates and 0.32, 1.0, 3.2, 10 and 32 mg/L loading rate WAF.

Definitive Test: 10, 18, 32, 56 and 100 mg/L loading rates (Nominal)
Chemical analysis of the 56 and 100 mg/L loading rate WAF fresh test preparations at 0 hours showed measured test concentrations of 1.38 and 0.900 mg/L, respectively. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations of 2.59 and 0.979 mg/L, respectively.
Details on test conditions:
Experimental Design and Study Conduct
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Preliminary solubility work indicated by visual observation that not all of the undissolved test item could be removed following filtration through a glass wool plug and filter paper. Therefore, any immobilised daphnia in the initial experiments and definitive test were examined microscopically in order to determine whether the effects seen were physiological rather than toxicological.

Range Finding Test
The loading rates to be used in the first initial experiment were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
In the range-finding test, 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 20 °C to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours, the number of immobilized daphnids was recorded.
The control group was maintained under identical conditions, but not exposed to the test item.

Initial Experiments
Based on the results of the range-finding test, an initial experiment was conducted at a test series range of 10, 18, 32, 56 and 100 mg/L loading rate WAF.
Due to high immobilization in all loading rates, a second experiment was performed at a test series range of 0.32, 1.0, 3.2, 10 and 32 mg/L loading rate WAF.
Insufficient immobilization was observed in the second experiment to allow the calculation of an EL50 value. The significant immobilization observed in the initial experiment was considered to be attributable to the presence of undissolved test item following filtration. Following microscopic observations, filtration in the second experiment was considered to have removed the dispersed test item.

Definitve Test
Based on the results of the range-finding test and initial experiments, the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Exposure Conditions
As in the range-finding test, 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions, but not exposed to the test item.
The test preparations were not renewed during the exposure period.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
56 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
100 other: mg/L Loading Rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at 1.0 and 10 mg/L loading rate WAF; however, immobilization was observed at 100 mg/L loading rate WAF.
Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L were selected for the initial experiment.

Initial Experiments
In the initial experiment, there was 55% immobilization in the 10 mg/L loading rate WAF and 100% immobilization in the 18 to 100 mg/L loading rates at 48 hours. Consequently, a NOEL and EL50 were not able to be identified.
The second experiment had no immobilization in the 0.32, 1.0 and 10 mg/L loading rate WAFs; however, there was immobilization in the 3.2 mg/L loading rate (20%) and 32 mg/L loading rate (5%).
Review of the study data indicated that the toxicity observed in the initial experiment may be due to the presence of undissolved components of the test item. In both experiments, microscopic observations of the immobilized daphnia found no test item adhered to the daphnia, indicating a toxicological response to the test item rather than a physiological one.
For the definitive test, the original series of 10, 18, 32, 56 and 100 mg/L loading rate WAF was used, and no change was made to the experimental procedure.

Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the 56 and 100 mg/L loading rate WAF fresh test preparations at 0 hours showed measured test concentrations of 1.38 and 0.900 mg/L, respectively. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations of 2.59 and 0.979 mg/L, respectively.
As the 56 mg/L loading rate WAF measured concentrations were greater than the 100 mg/L loading rate WAF measured concentrations, and the 0 and 48 hour results appeared to be switched, the frozen duplicate samples were analyzed to confirm the results.
Chemical analysis of the frozen duplicate samples of the 56 and 100 mg/L loading rate WAF fresh test preparations at 0 hours showed measured test concentrations of 2.43 and 0.996 mg/L, respectively. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations of 1.46 and 1.08 mg/L, respectively.
The frozen duplicate samples of the 100 mg/L loading rate WAF were analyzed a second time to determine if there was an effect from dilution during sample preparation on measured concentrations. The sample preparation factor was changed from 4.0 to 2.0, which was the same as used for analysis of the control and 56 mg/L loading rate WAF samples. The measured concentrations at 0 and 48 hours were 0.942 and 1.02, respectively, and the dilution method was considered to not have an effect on the measured concentration.
The chemical analysis reported is the measured concentrations of the frozen duplicate samples.
The difference in measured concentrations between the 56 and 100 mg/L loading rate was considered to be due to the surfactant nature of the test item. The test item was insoluble in test water and the analytical method measured a component of the test item that was soluble. The chemistry of the test item suggests that test item in solution would accumulate and visual observations during the study indicated that undissolved test item was not always removed through the WAF filtration process. The loading rates were considered to have been prepared correctly, as was indicated by the biological response; however, by the nature of the test item accurate analytical measurements were not possible.
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 'Cummulative Immobilization Data and Observations in the Definitive Test'.
Microscopic observations performed on immobilized daphnia indicated that no visible test item was adhered to the daphnia. Therefore, immobilization was considered to have been due to toxicological effects from the test item rather than a physiological effect.
Analysis of the immobilization data by visual inspection at 24 hours and by Probit analysis using Linear Maximum-Likelihood regression at 48 hours, based on the nominal loading rates, gave the following results:

At 24 hours: EL50 (mg/L Loading Rate WAF), >100. 95% Confidence limits (mg/L Loading Rate WAF), not determined
At 48 hours: EL50 (mg/L Loading Rate WAF), >100. 95% Confidence limits (mg/L Loading Rate WAF), not determined
The No Observed Effect Loading rates after 24 and 48 hours exposure were 100 and 56 mg/L loading rate WAF, respectively. Correspondingly, the Lowest Effect Loading rate after 48 hours of exposure was considered to be 100 mg/L loading rate WAF.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
Temperature was maintained at 21 ºC to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 492 to 536 lux.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 10, 18, 32, 56, and 100 mg/L loading rates were observed to be a clear, colorless water column with test item adhered to a glass slide suspended in the center and small amounts of test item floating throughout.
After 23 hours stirring and a 1-Hour standing period, the 10 mg/L loading rate was observed to be very slightly cloudy. The 18 mg/L loading rate was slightly cloudy, and the glass slide was observed to have fallen from suspension during preparation. The 32 mg/L loading rate was observed to be cloudy with visible lumps of test item and test item on the surface. The 56 and 100 mg/L loading rates were observed to be very cloudy and very very cloudy, respectively.
Visual examination of the WAFs showed test item present and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length) and a piece of filter paper. There was no dispersed test item observed by microscopic examination after filtering through the glass wool plug and filter paper.
During the test, the control and all loading rates were observed to be clear, colorless solutions


Results with reference substance (positive control):
A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

Time point 24 hours: EC50 (mg/L), 1.3. 95% Confidence Limits (mg/L), 1.0 - 1.8. No Observed Effect Concentration (NOEC) (mg/L), 1.0. Lowest Observed Effect Concentration (LOEC) (mg/L), 1.8.
Time point 48 hours: EC50 (mg/L), 1.2. 95% Confidence Limits (mg/L), 1.1 - 1.3. No Observed Effect Concentration (NOEC) (mg/L), 0.56. Lowest Observed Effect Concentration (LOEC) (mg/L), 1.0.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Table           Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Loading Rate


(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

Nominal
Loading Rate


(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

0

0

1

0

1

5

5 N

5 N

4 N

5 N

100

3

2

1

2

8

40

2 N

3 N

4 N

3 N

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:
Time Point: 48 hours
EL50 (mg/L Loading Rate WAF): >100
No Observed Effect Loading Rate (NOEL) (mg/L): 56
Lowest Observed Effect Loading Rate (LOEL) (mg/L): 100


Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test and initial experiments, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the 56 and 100 mg/L loading rate WAF fresh test preparations at 0 hours showed measured test concentrations of 2.43 and 0.996 mg/L, respectively. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations of 1.46 and 1.08 mg/L, respectively.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item gave the following results:

Time Point

EL50

No Observed Effect Loading Rate

(NOEL)

Lowest Observed Effect Loading Rate

(LOEL)

(Hours)

(mg/L Loading Rate WAF)

(mg/L)

(mg/L)

48

> 100

56

100

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