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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
other: stability study under OECD 222 conditions without test organism
Adequacy of study:
supporting study
Study period:
15 April - 11 June 2015
Reliability:
other: not applicable
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
stability test under OECD 222 conditions without addition of test organism
Analytical monitoring:
yes
Details on sampling:
Experiment 1: Initial recovery.
Each jar was sampled in four separate locations by placing the jar on its side and taking soil from the top, middle sides and bottom of the jar.
Soil samples were extracted twice with THF and the resulting extracts counted using LSC and analyzed by HPLC with radiometric detection (HPLC-RAD).

Experiment 2: Homogeneity.
The jar was removed from the jar mill and tapped to dislodge soil caked onto the jar side.
The jar was sampled in four separate locations by placing the jar on its side and taking soil from the top, two samples from the middle and bottom of the jar.

Experiment 3: Stability.
All five beakers were sampled on Day 1.
Three beakers were sampled each on Day 2 and Day 3, with each beaker being sampled multiple times.
Beakers were core sampled using a 5 mL syringe (with the end removed).
The core sample was extruded from the syringe and divided into a top and bottom sample.
Each sample was solvent extracted as outlined below and analyzed by LSC and HPLC-RAD.
After coring, the beaker was gently shaken to allow the hole created by the core to collapse in on itself to maintain a constant exposed surface area for potential volatilisation.
Vehicle:
no
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Details on test conditions:
TEST SYSTEM
-Due to the high vapor pressure of the test article, volatilisation was anticipated to be a significant source of loss.

Experiment 1: Initial recovery. The effect of varying amounts of headspace in the mixing vessel on initial recovery.
Three 250 mL safety coated jars with polytetrafluoroethylene (PTFE) lined lids.
The jars were filled with varying amounts of dry soil.
Water was added to bring moisture to approximately 48% of the water holding capacity.
After taping the lid closed to avoid inadvertent opening, the jars were placed on a US Stoneware Jar Mill (model 764) and rolled for 24 hours at a slow setting to allow the soil to tumble.
The jars were occasionally gently tapped to remove any caking of the soil on the sides of the jar.
Three - 10 uL Hamilton gastight syringes were fitted with Cheney adaptors and were calibrated to deliver a known of mass L3.
Each jar was spiked with 14C-L3 for a final target concentration of 32.9 mg 14C-L3 / kg soil dw, and returned to the jar mill roller to equilibrate overnight.
Each jar was removed from the jar mill and tapped to dislodge soil caked onto the jar side.
Each jar was sampled in four separate locations by placing the jar on its side and taking soil from the top, middle sides and bottom of the jar.
Soil samples were extracted twice with THF and the resulting extracts counted using LSC and analyzed by HPLC with radiometric detection (HPLC-RAD).

Experiment 2: Homogeneity.
1000 mL wide mouth glass jar with a polytetra-fluoroethylene (PTFE) lined lid.
The threads of the jar were covered with PTFE tape to reduce volatilisation of the test article.
The test soil was filtered with a screen prior to use.
The jar was filled approximately 9/10 full with about 590 g (d.w.) of the test soil (2014 100 Rochester Loam received March 2015).
The soil was brought to a target moisture concentration of 18.8% w/w, or approximately 48% of the measured water holding capacity (WHC).
The jar was placed on a US Stoneware Jar Mill (model 764) and rolled for 24 hours at a slow setting to allow the soil to tumble.
Using a calibrated gastight syringe fitted with a Cheney Adaptor, the soil was then spiked with 26.0 mg of 14C-L3. This concentration was 34% above the saturation concentration, to attempt to account for the losses seen during the initial recovery experiment.
After taping the lid closed to avoid inadvertent opening, the jar was place on the jar mill and rolled for 18 hours at a slow setting to allow the soil to tumble.
The jar was occasionally gently tapped to remove any caking of the soil on the sides of the jar.
The jar was removed from the jar mill and tapped to dislodge soil caked onto the jar side.
The jar was sampled in four separate locations by placing the jar on its side and taking soil from the top, two samples from the middle and bottom of the jar.

Experiment 3: Stability.
The spiked soil from the homogeneity experiment was divided into five beakers.
The 100 mL beakers used were Kimax® tall form beakers (internal dimensions 4.5 cm wide and 7.2 cm tall, 120 mL capacity) without a pour spout.
The loose soil volume was approximately 110 mL, initially leaving about 10 mL of headspace.
Each beaker was covered with plastic film having five small holes (approximately 3mm in diameter) to allow for air exchange.
The plastic film was secured with rubber bands.
The moisture content was monitored gravimetrically and additional Milli Q water was added, as needed.
The average of the homogeneity determination was taken as the initial concentration for each beaker.
All five beakers were sampled on Day 1.
Three beakers were sampled each on Day 2 and Day 3, with each beaker being sampled multiple times.
Beakers were core sampled using a 5 mL syringe (with the end removed).
The core sample was extruded from the syringe and divided into a top and bottom sample.
Each sample was solvent extracted as outlined below and analyzed by LSC and HPLC-RAD.
After coring, the beaker was gently shaken to allow the hole created by the core to collapse in on itself to maintain a constant exposed surface area for potential volatilisation.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Rochester loam obtained from Smithers Viscient (Wareham, MA).
- Pesticide use history at the collection site: Not reported
- Collection procedures: Not reported
- Sampling depth (cm): Not reported
- Soil texture (if natural soil)
- % sand: 77
- % silt: 14
- % clay: 9
- Soil taxonomic classification: Sandy loam
- Soil classification system: USDA Textural Class
- Organic carbon (%): 4.36
- Maximum water holding capacity (in % dry weight):39.7
- CEC: 10.8
- Pretreatment of soil: Not reported
- Storage (condition, duration): Not reported
- Stability and homogeneity of test material in the medium: See results
Reported statistics and error estimates:
Numerical data obtained during the study were processed using Microsoft Excel™ 2013. Average and % relative standard deviation were calculated where noted.

Initial Recovery: Based on the results of the initial recovery experiment, the main experiment test vessel was selected in order to minimize headspace as much as possible, while still allowing for enough tumbling to ensure homogeneity. However, even the container with the best recovery only observed an average recovery of 69.7% (or 22.9 mg/kg soil (d.w.)). For this reason, the final experiment was “over spiked” by 34% to compensate for initial losses due to volatilization. Although samples were not combusted for the initial recovery experiment, another experiment showed that combusted samples contributed minimally as a percentage of the overall observed radioactivity, averaging 3.6% of observed radioactivity.

Table 1. Results of initial recovery experiment:

Soil Added (d.w.), g

Jar Volume, mL

Expected DPM

Average Observed, DPM

Average Recovery, %

% RSD

144

250

474971

330829

69.7

8.7

106

250

486349

246428

50.7

14.7

68

250

471968

 319340

67.7

8.2

Homogeneity: The 14C-L3 was spiked at a concentration greater than the theoretical solubility of L3 in soil based on its organic carbon content, in order to attempt to compensate for initial losses noted in the initial recovery experiment. Even though the spiked amount was increased by 34% to account for initial losses, the initial average achieved of 23.1 mg/kg soil (d.w.) is lower than the target concentration of 32.9 mg/kg soil (d.w.), and consistent with the maximum recovery achieved in the initial recovery experiment. This could be due to the maximum sorption capacity being slightly lower than the theoretically calculated value. The extracts analysed by HPLC-RAD showed a single peak consistent with the parent. The counts from the LSC showed a slightly higher concentration at the top of the sample jar than the bottom, with an average DPM/g equating to an initial concentration of 23 mg 14C-L3 / kg soil (d.w.), and a relative standard deviation of 16.9%. The results of the homogeneity are shown in the following table. Table 2. Results of Homogeneity Experiment
 Sample location  DPM/g (d.w.)
 Top  343676
 Middle Top 279010 
 Middle Bottom  244586
 Bottom  243736
 Average  277752
 % RSD  16.9

Extraction Efficiency: All samples, as described above were extracted twice with THF. In order to verify that most of the test article was extracted in these first two extractions, a separate third extraction was performed on samples prepared similarly to the homogeneity samples. An average of 4.0% of the expected radioactivity was found in the third extraction. After the third extraction, the remaining soil was combusted in the biological oxidiser and the resulting solution analysed by LSC. These samples showed an additional 3.6% of the expected radioactivity remained, indicating that 7.6% of the expected radioactivity was not extracted by the first two THF extractions. This equates to an extraction efficiency of 92.4%. Stability: The average of the initial homogeneity was taken to be the initial concentration in the beakers for Day 0. Samples taken on Days 1, 2 and 3 showed significant losses of parent. Profiling by HLPC-RAD showed a single peak (where observed) consistent with parent in all but one chromatogram. Day 1 Beaker D, bottom aliquot, showed approximately 22% of remaining activity as a peak consistent with the degradation product dimethylsilanediol. Dimethylsilanediol was not observed in any other samples, including Day 1 Beaker D top aliquot. Due to significant losses of parent, sampling was stopped on Day 3. Table 3: Remaining Radioactivity of each Beaker during Stability Experiment

 

DPM/g (d.w.)

Date

5/18/2015

5/19/2015

5/20/2015

5/21/2015

Day

0

1

2

3

Beaker A

277752

60585

32942

23144

Beaker B

277752

65464

33038

15789

Beaker C

277752

56504

34750

14678

(13591)*

Beaker D

277752

58837

Beaker E

277752

61319

Average % of Initial Remaining

100%

21.8%

12.1%

6.3%

*Second extraction vial broke in the centrifuge.  Value reported is representative of the top extract aliquot.  Value in parentheses is the actual average of the top and bottom aliquots, but should not be used due to the loss in the second extract.

Conclusions:
The homogeneity experiment was spiked with 14C-L3 to achieve an initial concentration of 23 mg 14C-L3 / kg soil (d.w.) (70% of target) and a relative standard deviation of 16.9%. The stability experiment showed significant losses of 14C-L3 over the first few days of the experiment. By day 3, only 6.3% of the initially observed radioactivity was detected, and sampling was stopped. Profiling by HPLC-RAD showed a single peak (where observed) consistent with 14C-L3 in all but one chromatogram. The absence of degradation products in the vast majority of samples, coupled with the rapid loss of 14C activity, shows that the primary mechanism of test article loss was volatilization of 14C-L3 from the simulated OECD 222 test setup.

Description of key information

Key value for chemical safety assessment

Additional information

There are no reliable data describing the long-term toxicity of the registered substance to soil macroorganisms.

Stability study using the related substance octamethyltrisiloxane (L3) under OECD TG 222 conditions without test organisms:

A stability/recovery test was conducted in preparation for terrestrial ecotoxicology studies with the related substance L3. Vi4D4 and L3 are members of the Siloxane Category of compounds. Both substances have an air-soil partition coefficient >1. In addition, the substances have a slow hydrolysis rate relative to the time-scale of ecotoxicity testing (t1/2 = 63 h and 329 h at pH 7 and 25°C). In the context of terrestrial toxicity, both L3 and Vi4D4 are expected to have similar stability in soil and prior to volatilisation, any exposure is likely to be to the parent substance during the terrestrial toxicity studies. Therefore it is considered valid to read-across the results of the soil stability study with L3.

The study demonstrated a method of introducing neat 14C-octamethyltrisiloxane (14C-L3) into natural soil with subsequent mixing to distribute the test article throughout the soil uniformly.

The second phase of the study investigated the stability of 14C-L3 in the same soil under conditions representative of those used for the OECD TG 222 Earthworm Acute Toxicity and Reproduction Test. The system was partially open to allow for respiration during a planned future toxicity experiment.

Effect of headspace on loss of test material during dosing of soil was examined. The main experiment test vessel was selected in order to minimise headspace as much as possible, while still allowing for enough tumbling to ensure homogeneity. However, even the container with the best recovery only observed an average recovery of 69.7% (or 22.9 mg/kg soil (d.w.)).

Measures were taken to avoid loss of test substance through volatilisation during the homogeneity experiment. These included:

- fitting the test vessel jar with a polytetrafluoroethylene (PTFE) lined lid and covering the threads of the jar with PTFE tape;

- spiking the soil with a calibrated gastight syringe;

- spiking with a concentration 34% above the saturation concentration, to attempt to account for test substance losses;

- taping the lid closed to avoid inadvertent opening.

The stability experiment was then carried out using the spiked soil from the homogeneity experiment, which was divided into five beakers. Each beaker had 10 mL of headspace and was covered with plastic film having five small holes (approximately 3 mm in diameter) to allow for air exchange. The plastic film was secured with rubber bands.

By day 3 in the stability experiment, only 6.3% of the initially observed radioactivity was detected, and sampling was stopped. However, the average of the homogeneity determination was taken as the initial concentration for each beaker in the stability test. The soil was not analysed again after dividing into the beakers, therefore the potential loss from moving the test soil from the container used in the homogeneity experiment to the 5 test beakers used in the stability study was not determined.

A single peak was observed consistent with 14C-L3, showing that the primary mechanism of loss in the initial recovery experiment was volatilisation.

The absence of degradation products in the vast majority of samples, coupled with the rapid loss of 14C activity, shows that the primary mechanism of test article loss was volatilisation of 14C-L3 from the simulated OECD TG 222 test setup.

Vi4-D4 has a higher air-soil partition coefficient that L3, meaning that similar losses from the test system would be expected under equivalent test conditions. Additionally, in an OECD TG 216 (Soil Microorganisms: Nitrogen Transformation Test) study for the effects of the related substances L2 and L3 on nitrate formation rate of soil microflora, analysis of the test substance concentrations show that test material was lost by day three of the test (see Section 6.3.4). Based on these experimental findings, the registrants believe it is not technically feasible to conduct an OECD TG 222 test for the registration substance on the basis that the test substance is too volatile to maintain adequate concentrations in the test system.