Cyclohexylidenebis[tert-butyl] peroxide

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

A preliminary non-GLP study on the stability of the test item in aqueous medium without fish in a flow-through system was performed. In this study, it was determined that stable concentrations of the applied test concentration can be achieved. Therefore, it was considered that the BCF test can be performed as an aqueous application in a flow-through system. As the OECD 210 study was also running at the same time at the same testing facility, it was decided to wait the finalisation of the FELS test to determine which concentration will be the most suitable to perform a BCF pre-test and avoid any toxicity or adverse effects.
A BCF pre-test with nominal concentration of 50 µg/L (measured concentration ca. 25 µg/L) in a flow-through system with zebrafish was performed. The uptake phase was 28 days. During the uptake phase it was evident that the steady state required for a valid BCF test cannot be achieved. The test item was evaporating from the system despite the taken measures. The measured concentrations were fluctuating below and above the allowed ± 20 % range of the time weighted average measured concentration. Nevertheless, a depuration phase of 7 days was applied and a preliminary BCF above 5000 was calculated based on the unstable concentrations. Increase or decrease in the application of the test solution led to spikes or dips in the TWA concentration.
Based on this pre-test and the experience obtained during the FELS test, it was concluded that the determination of the bioaccumulation of the test item cannot be determined via the aqueous application route and the main study has to be performed via a dietary application.
For the BMF test a new method validation in feed needs to be evaluated, the appropriate test concentrations have to determined and a new pre-test incl. establishment of an adapted analytical method in feed will have to be performed before the main test. Therefore, the submission of the complete study on Bioaccumulation with the test item will have to be submitted at a later time. For more details please refer to the attached statement from the testing facility and the supporting study record on the BCF pre-test.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May to June 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

GLP compliance:

no

Radiolabelling:

no

Details on sampling:

- Fish sampling: 3 fish replicates per sampling
- Sampling schedule: 5 sampling - 3 throughout the uptake phase and 2 throughout the depuration phase
- uptake phase: day 14, 21, 28
- depuration phase: day 1, 7
- Water concentration sampling: 3 times per week

Vehicle:

yes

Remarks:

acetone

Details on preparation of test solutions, spiked fish food or sediment:

- Solvent content: 0.001%

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: based on initial stability test in aquatic medium without fish

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

7 d

Test temperature:

15 ± 2 °C

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type: covered
- Material, size, headspace, fill volume: glass, fill volume: 85 L
- Renewal rate of test solution (frequency/flow rate): 10-fold exchange of the waterbody (85 L) per day

Nominal and measured concentrations:

Nominal concentration: 50 µg/L
Measured concentration: 23.3 µg/L (TWA) (± 20% of TWA: 18.7 - 28.0 µg/L)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The BCF was estimated based on a non-steady state test item concentration and is not considered to be representative of the test items properties. Due to the high fluctuation of the test item concentration over the uptake phase, no reliable BCF can be derived.

Conc. / dose:

23.3 µg/L

Type:

BCF

Value:

> 5 000 dimensionless

Details on results:

No adverse effects in fish were observed.

Test concentration of the test item

Non-stable application within the range of the ±20 % range of the TWA (equilibration for 5 days before start of application) increasing fluctuation over time were observed. Readjustments of the application (syringe flow) were made to readjust system during test at 4 timepoints.

Kinetics of the test item in fish

Uptake phase: A steady state, as per definition in the OECD guideline, was not reached. As by worst case scenario, a mean concentration of 842 mg/kg was determined at the end of uptake (highest measured value). A BCF end uptake of 36072 was calculated by considering the C(fish d28/TWA).

Depuration phase: Based on the slope of the depuration (ln transformed data), a depuration rate constant was calculated with k2 = 0.188
A linear modelling with the data from the uptake phase ratios (fish to TWA concentration ratio) together with the depuration rate constant resulted in an uptake rate constant k1 = 5608.
A kinetic BCFk of 29907 was calculated by the ratio of k1/k2.
Calculation of half life based on k2 : t50% = 3.70 days ; t95% = 15.98 days.

Conclusions:

Steady state could not be reached, therefore, no steady state BCF could be correctly derived. A clear indication that a BMF study is needed was observed.

Executive summary:

Validity criterion of a stable water exposure for BCF GLP-test may not be met, as application becomes increasingly unstable and suffice not range of ±20 % TWA.
No equilibrium (i.e. steady state) was reached between fish and water concentration after 28 days (probably also due to the high variation in the test medium concentration) and “worst case” BCF (based on last sampling of uptake) was higher than BCFk.
As BCF calculated from all samplings and medium TWA was close to the kinetic BCFk, the exposure time was nevertheless close to being sufficient for reaching a steady state (both values should match, then exposure time chosen is sufficient).
Clear recommendation to perform a BMF study.

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

February - March 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Principles of method if other than guideline:

The suitability of the aqueous exposure of the test item for the performance of a fish bioconcentration study with rainbow trout was tested in the test facility of Fraunhofer IME, Germany.
OECD TG 305 does not generally recommend the use of solvents and dispersants (solubilizing agents). Nevertheless, due to the difficult physico-chemical properties of the test item, a solvent-mediated application was tested.

GLP compliance:

no

Radiolabelling:

no

Details on sampling:

- Sampling of aqueous test solutions: 7 mL of the aqueous samples were taken and extracted with 0.5 mL of n-hexane by shaking on a horizontal shaker for 30 min and subsequently transferring 200 µL of the organic phase to a micro vial for analysis.

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

- Holding and dilution water:
De-chlorinated local tap water was used for the application in accordance with the OECD TG 305. The tap water is sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process occurs on-site at Fraunhofer IME and includes filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation. To avoid copper contamination, plastic water pipes are used in the test facilities. The quality of the water is monitored monthly to assure that no contaminants affect the conducted studies (e.g. heavy metal concentrations below thresholds as specified in OECD TG 305).
- Preparation of test item stock solution:
A sufficiently concentrated solvent stock solution was prepared three times during the test.
The prepared stock solutions were diluted by a factor of 10 000 to obtain the final nominal test concentration of 50 µg/L with the maximal allowed amount of 0.01 % of solvent (acetone) in the test medium. The dilution was performed by filling the respective solvent stock solution in a 50 mL-syringe as a daily reservoir for the application (including some buffer in terms of volume to ensure that the application will not stop after 24 h). From there the eluate was combined with Cu-red tap water in a mixing chamber before entering the test vessel. For the final setting with the 10-fold flow rate the proportion of used solvent was reduced to 0.005 %.

Test organisms (species):

other: none

Details on test organisms:

No fish were exposed to the tets iem in this test. A stability test only with the test item were perfomed.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Test temperature:

13.3 - 13.8 °C

pH:

7.62 - 8.11

Dissolved oxygen:

96.9 - 115 % oxygen saturation

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type: covered with Acrylic glass shields to diminish evaporation
- Material, size, headspace, fill volume: glass, 100 L, approx. 90 L test medium
- Type of flow-through: metering pump system supplied with fresh dilution water, which was combined with a flow of test item dissolved in solvent from a syringe pump.
- Renewal rate of test solution (frequency/flow rate): Different flow rates were tested over the course of 3 weeks to evaluate in which setting the losses of test item due to volatility can be diminished. The performed flow rates were 5-fold in the first week, 2.5-fold in the second week and 10-fold in the third week.

Nominal and measured concentrations:

Nominal concentration: 50 µg/L

Reference substance (positive control):

no

Concentration of the test item in test medium
The application was run with a 5-fold exchange of the water body in the first week. The aimed nominal concentration in the test vessel was 50 µg/L. As the mean measured concentration in the test vessel was 18.2 µg/L and therefore much lower than expected (30.4 - 41.0 % from the nominal), the volatile character of the test item was suspected to be the cause of the low concentration. As it is likely that volatile test item is lost at the water surface of the test vessel, the concentration in the mixing chamber was also monitored from day 7 on. Here, nearly double the concentration with a mean of 38.6 µg/L, corresponding to recoveries of 72.2 - 82.2 % in relation to the nominal concentration was measured.
As high flow rates are known to promote volatilization, a lower flow rate was used in the second week. The test system was thus adjusted to operate with a 2.5-fold exchange of the water body per day. The mean concentration in the vessel was 10.8 µg/L (19.9 - 24.9 % from the nominal), in the mixing chamber a mean of 29.5 µg/L was determined (43.4 -70.3 % from the nominal). In addition, it became obvious that the %-recoveries in mixing chamber were decreasing over time of the exposure. This effect coincided with the observation of a biofilm formation in the test vessel during the second week, which may be caused by the use of solvent. Since an increased biofilm affects also the availability of the test item in water, the system was stopped, disassembled and cleaned before further experimentation.
Since the so-far tested setups were not sufficient to achieve the desired test concentration, a high flow with a 10-fold exchange of the water body per day was tested in the third week. Here the amount of solvent used for the application was reduced to 0.005 % to diminish the biofilm growth in the system. The resulting mean concentration in the mixing chamber was 41.6 µg/L (with recoveries from 76.7 - 87.9 % from the nominal) and 27.7 µg/L in the test vessel (corresponding to recoveries of 54.2 - 57.6 % from the nominal).

 

Table 1: Solvent-facilitated application: medium concentrations of the test item in test vessel and mixing chamber

Exchange rate	Exposure day	Conc. In vessel	Mean conc. In vessel [µg/L]	RSD from mean [%]	[%] From Nominal	Conc. In mix. ch. [µg/L]	Mean conc. In vessel [µg/L]	RSD from mean [%]	[%] From Nominal
5-fold	3	17.6	18.2	12.8	35.3	NA	38.6	9.2	NA
	5	15.2			30.4	NA			NA
	7	19.5			39.1	41.1			82.2
	10	20.5			41.0	36.1			72.2
2.5-fold	12	12.5	10.8	13.4	24.9	35.2	29.5	23.6	70.3
	14	10.5			20.0	31.5			63.0
	17	9.93			19.9	21.7			43.4
The test system was stopped and disassembled. All surfaces were cleaned before start with 10-fold exchange rate.
10-fold	2	27.1	27.7	3.59	54.2	42.5	41.6	6.98	85.0
	4	28.8			57.6	44.0			87.9
	7	27.1			54.2	38.4			76.7

Conclusions:

Investigations were carried out to find an appropriate setup for an aqueous exposure for a bioconcentration study according to OECD TG 305 with the test item.
The analysed concentrations of test item measured in the aqueous solutions showed that a stable concentration can be achieved with the use of solvent combined with a high flow rate. For a valid BCF-test, the concentration applied in water shall be at a stable level with ± 20 % RSD. However, the application via solvent (acetone) also turned out to promote a growth of biofilm in the mixing chamber and test vessel that diminishes the concentration of the test item. Thus, an exchange of the test system components shall be performed at least every 10 days during the exposure phase.
The circumstance of substance-loss from the water phase due to the volatile character of the test item cannot be ruled out during testing. It became visible, that during the application with the high flow rate the highest recoveries were achieved among the tested settings. Nevertheless, approx. 40 % of the applied test item could not be recovered from the test water. With regard to the toxic properties of the test item, the performance of the test shall be accomplished only in a setup which ensures that evaporated test item is aspirated by an appropriate flue-system.

Description of key information

Due to analytical and test design issues, the main test on bioaccumulation had to be posponed. For more details refer to the study records in this endpoint. The study will be submitted in a dossier update as soon as it is finalised and the chemical safety assessment has been updated.