[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: GLP Guideline study

Justification for type of information:

see read across rationale in Section 13.2

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Chemical analysis was conducted on the sediment, overlying water and interstitial water at test initiation (Day 0) and termination (Day 28).

One additional test vessel was prepared for each treatment group, for destructive sampling at Day 0, post-equilibration phase. At test termination, one of the six remaining vessels was also destructively sampled as described below. Destructive sampling was not conducted for the untreated control. Destructive sampling involved separation of overlying water, interstitial water (sediment pore water) and sediment, followed by chemical analysis of all three samples. Destructive sampling was conducted by carefully decanting the overlying water without disrupting the sediment. The volume of overlying water was recorded. Interstitial water was separated from the sediment solids by transferring the entire sediment sample to individual pre-weighed centrifuge pots and centrifuging at 2500 g (Multifuge x3r Centrifuge) for 30 min. The supernatant volume was recorded. The remaining sediment solids in centrifuge pots were weighed and then transferred into a plastic container. The sediment was then thoroughly mixed by hand and duplicate samples (ca 10 g) were removed and added to pre-weighed beakers to determine the moisture content.

The remaining sediment, the overlying water and interstitial water was then analysed using the analytical methods validated under Charles River Study 223110.

Vehicle:

yes

Details on sediment and application:

Artificial sediment was prepared for both the range finding and definitive tests with the following approximate composition on a percent w/w basis (dry weight):
Air dried, ground peat (100% sphagnum moss peat, J. Arthurs Bowers, Strawberry Corner, Edinburgh): 5 % w/w
Kaolin clay (ECC International Limited, St. Austell, UK): 20 % w/w
Fine silica sand (Mineral Marketing, UK): 75 % w/w

A peat suspension was prepared for each treatment and control group by mixing the required amounts of the air-dried, ground peat with HPLC grade water (at a volume equivalent to 40% moisture content in the final sediment). The peat suspension was conditioned for 7 days with continuous mixing, to allow the stabilisation of the pH and establish a stable microbial composition.

For the range-finding test, a 100 mg/mL acetone stock solution was prepared for the 1000 mg/kg treatment by dissolving a weighed amount (500.20 mg) of test item in 5 mL of acetone. A 1:4 serial dilution was then used to prepare the 1, 10 and 100 mg/kg stock solutions. Acetone or test item stock solution (3 mL) was added to individual portions of sand (ca 20 g retained from the 75% final amount). The treated sand portions were then placed under the fume hood until the solvent had completely volatilised. The remaining dry constituents were combined with the peat suspension in a Breville 1HBO86 blender. The sediment was mixed for ca 30 secs until homogenous. The treated sand was combined with the homogenous sediment and mixed for a further ca 30 secs. No test item or solvent was added to the control sand. The sediment was adjusted to a final pH of 6.5-7.5 using calcium carbonate. A sediment:water depth ratio of 1:3 was prepared for each vessel. This ratio used is an error but was has been considered to have no effect on the validity or outcome of the study. Treated or control sediment was added to each beaker, to a depth of ca 3 cm (mean wet weight = ca 288g). Reconstituted freshwater, to a depth of ca 9 cm above the sediment, was added to each vessel with minimal disruption to the sediment. After preparation, the test vessels were equilibrated for 7 days.

For the definitive test, a 250 mg/mL acetone stock solution was prepared for the 1000 mg/kg treatment by dissolving a weighed amount (2500.36 mg) of test item in 10 mL of acetone. A 10 mL stock of untreated acetone only was prepared for the solvent control. Acetone or Test item stock solution (10 mL) was added to individual portions of sand (ca 200 g retained from the 75% final amount). The treated sand portions were then placed under the fume hood overnight until the solvent had completely volatilised. Required amounts of peat suspension, clay and sand were transferred to an electric mixer (Hobart Industrial mixer) and mixed with the test item (or acetone) spiked sand. No test item or solvent was added to the control sand. The sediment was adjusted to a final pH of 6.5-7.5 using calcium carbonate. A sediment:water depth ratio of 1:4 was prepared for each vessel. Treated or control sediment was added to each beaker, to a depth of ca 3 cm (mean wet weight = ca 364 g). Reconstituted freshwater, to a depth of ca 12 cm above the sediment, was added to each vessel with minimal disruption to the sediment. After preparation, the test vessels were equilibrated for ca 48 h.

Test organisms (species):

Chironomus riparius

Details on test organisms:

The test species are the common non-biting freshwater dipteran Chironomus riparius Meigen (Diptera: Chironomidea). The test species was cultured in-house from a culture originating from Aquatic Research Organisms, P.O. Box 1271, One Lafayette Road, Hampton, NH 03843, USA.

During the study, a volume of Tetramin® feed suspension, equivalent to 0.5 mg/chironomid/day was added in the first 10 days and 1.0 mg/chironomid/day for the remainder of the exposure period. Green algae, Chorella vulgaris, were added on Day 0 to supplement this diet. The feed suspension was applied to the water surface.

Synchronous chironomid cultures have been prepared for the production of first instar larvae (2-3 days post hatch) for use in the range finding and definitive tests.

After the equilibration period, 20 first instar C. riparius larvae were added to each vessel. Aeration was started 24 h after addition of the larvae.

The number and sex of emerged adult chironomids was recorded daily. Emerged adults were removed from the test vessels.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

none

Hardness:

<400 mg CaCO3/L

Test temperature:

20 ± 2°C

pH:

initial range: 6 - 9

Dissolved oxygen:

> 60%

Salinity:

N/A

Nominal and measured concentrations:

The range finding test was conducted over 28 day period at the following nominal test item concentrations: 1, 10, 100 and 1000 mg/kg (dry weight sediment) together with an untreated control and solvent control. A single test vessel was prepared for each treatment group.

Based on the results of the range finding test, a limit test was conducted at a test item concentration of 1000 mg/kg, together with a solvent control and control over 28 days.

Seven vessels were prepared for each treatment group; six vessels for emergence observations and terminal chemical analysis and one vessel for chemical analysis on Day 0 (destructive samples). Only six vessels were prepared for the control group as no analysis was required.

Initial measured concentration: 893.69 mg/kg dry sediment

Details on test conditions:

Samples of excess control sediment were taken to determine the organic carbon content (TOC). Two aliquots of control sediment (ca. 13.5 g wet weight) were weighed and dried overnight in an oven at ca. 105°C. After cooling, the samples were weighed again.

During the equilibration phase and from 24 h post-addition of the larvae, vessels were aerated via a narrow bore glass tube to achieve a dissolved oxygen concentration ≥60% ASV. Test vessels were maintained within a temperature-controlled laboratory programmed to produce a constant temperature of 20±2°C, with a photo-period of 16 h light:8 h dark. Artificial daylight fluorescent tubes provided illumination in the laboratory (500-1000 lux). The water level in all vessels was monitored and replenished with HPLC grade water when necessary.

The pH of the sediment was taken prior to equilibration with reconstituted freshwater.

Actual temperature, dissolved oxygen concentration and pH was measured at the start of the test and then at weekly intervals in the overlying water of all vessels. Temperature was recorded daily via a digital max-min thermometer, which was placed in one control vessel.

Chlorine, hardness and conductivity of the reconstituted freshwater was measured during the conditioning period.

Dissolved oxygen was measured using a YSI 550A dissolved oxygen meter; temperature with a Hanna HI 93510 and digital max-min thermometer; pH with a Jenway 370 pH meter and conductivity with a Jenway 470 conductivity meter. The total hardness and chlorine were determined using a Hach Hardness test kit and a Hach chlorine test kit.

Light intensity was measured at bench level at the start of the test using a Lutron LX-101 lux meter.

The water used for the culturing and for addition to test vessels in the range-finding test, to generate “overlying water” was reconstituted fresh water prepared at Charles River with high grade salts and reverse osmosis grade water. The salts were dissolved in a volume of deionised water (500 L) and this solution was metered into a flow of deionised water, to produce reconstituted freshwater. For the limit test, reconstituted freshwater was prepared at Charles River, with high grade salts and HPLC grade water and was used during the holding and testing phases of the study.

All culture vessels were of glass construction and test vessels were 1 litre capacity “tall-form” glass beakers. They were aerated via a narrow bore glass tube. A layer of artificial sediment were placed in each vessel. The ratio of the depth of the sediment layer to the depth of the overlying water was 1:4. The top of each test vessel was covered with a fine mesh to confine newly-emerged adults.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

891.81 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Details on results:

During the range-finding test, no test item effect was observed at any of the treated concentration (1, 10, 100 and 1000 mg/kg (dry weight basis) when compared with the untreated control and solvent control.

During the limit test, the distribution data showed that the majority of the test item remained within the sediment. The mean measured concentrations at Day 0 and 28 showed that 89.18-95.62% of the test item was recovered from the sediment, 0.00% was found in the interstitial water and 0.00-0.01% was found in the overlying water. The mean measured concentrations for Day 0 and 28 were within 20% of the nominal test item concentration. T

During the limit test, the pH (7.29-8.38), temperature (control replicate max-min: 20.6-21.5°C, weekly actual: 20.2-21.7°C) and dissolved oxygen (61.5-100.0%) content of the overlying water remained within protocol specification. Light intensity was measured at bench level at the start of the test and was determined to be 620 lux. Chlorine, hardness and conductivity values of the reconstituted freshwater recorded during the range finding test were 0.01 mg/L, 235.2 mg CaCO3/L and 606 μS/cm, respectively.

Reported statistics and error estimates:

No statistical analysis was conducted for this test, because no negative effects (mean values in the treatment were higher than those in controls) on development time and total number of fully emerged male and female midges were observed.

 Mean Measured Concentration of Test Item During the Limit Test

Timepoint (Day)	Mean Measured Test Item* Concentration (mg/kg dry sediment)
	Sediment	Overlying Water	Interstitial Water	Total
0	891.81	0.026	0.004	891.84
28	956.16	0.045	0.066	956.27

* Test Item = [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tertbutyl] peroxide

  No test item was measured in the solvent control.

Distribution of Test Item between the Sediment, Overlying Water and Interstitial Water

Timepoint (Day)	Distribution of Test Item* (%)
	Sediment	Overlying Water	Interstitial Water
0	89.18	0.00	0.00
28	95.62	0.00	0.01

* Test Item = [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tertbutyl] peroxide

Validity criteria fulfilled:

yes

Remarks:

see details in Conclusions

Conclusions:

The test was considered valid under the criteria of OECD Guideline 218 as:
• The control adult emergence was 70% or greater in all replicates (control and solvent control emergence = 89% and 97%, respectively) and the majority of emergence occurred between Day 12 and 23 (emergence occurred between Day 14-24).
• The dissolved oxygen of all test replicates was greater than 60% (≥61.5%).
• The pH in all replicates remained in the pH range 6 to 9 (pH ranged from pH 7.29-8.38).
• The temperature range of the overlying water did not deviate by more than ±1°C during the test (control replicate max-min = 20.6-21.5°C, weekly actual = 20.2 21.7°C).

Executive summary:

The effects of prolonged exposure to [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was evaluated on the sediment-dwelling larvae of the fresh water dipteran Chironomus riparius Meigen (Diptera: Chironomidae) in a limit test performed in accordance with OECD testing guideline 218 and GLP requirements (Hugill, 2015).

The artificial sediment was fortified with [1,3(or 1,4)‑phenylenebis(1 -methylethylidene)] bis[tertbutyl] peroxide, at a nominal concentration of 1000 mg/kg dry weight basis (corresponding to 891.81 mg/kg dw expressed as initial measured concentration in s diment). The sediment was transferred to test vessels and covered with reconstituted water. First instar larvae were introduced into the sediment water system. The larvae were fed daily throughout the test. Exposure continued for 28 days, during which period the time, numbers and sex of emerging adults were recorded. The overlying water was gently aerated, but was not be renewed during the test (i.e.a static system). The validity criteria were fulfilled. The NOEC 28 days was calculated to be > 891.81 mg/kg dw (Day 0 measured sediment concentration) as no significant effect on the emergence of chironomids was observed at the tested concentration compared to the controls.

Description of key information

The effects of prolonged exposure to [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was evaluated on the sediment-dwelling larvae of the fresh water dipteran Chironomus riparius Meigen (Diptera: Chironomidae) in a limit test performed in accordance with OECD testing guideline 218 and GLP requirements (Hugill, 2015).

The artificial sediment was fortified with [1,3(or 1,4)‑phenylenebis(1 -methylethylidene)] bis[tertbutyl] peroxide, at a nominal concentration of 1000 mg/kg dry weight basis (corresponding to 891.81 mg/kg dw expressed as initial measured concentration). The sediment was transferred to test vessels and covered with reconstituted water. First instar larvae were introduced into the sediment water system. The larvae were fed daily throughout the test. Exposure continued for 28 days, during which period the time, numbers and sex of emerging adults were recorded. The overlying water was gently aerated, but was not be renewed during the test (i.e.a static system).The validity criteria were fulfilled. The NOEC 28 days was calculated to be > 891.81 mg/kg dw as no significant effect on the emergence of chironomids was observed at the tested concentration compared to the controls.