Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Testing was conducted between 29 July 2013 and 03 August 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Test Guidelines and to GLP.

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:

A slow stir preparation method or ( WAF/WSF) was used to allow preparation as close as possible to the water solubility of the test substance and
minimise dispersion of un-dissolved test material. The propensity of the test material to form dispersions has in previous studies resulted in
numerous cases of spurious physical effects on the test organisms. A complete description of the solution preparation methods are described in the test solutions section.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the each loading rate WAF test group (replicates R1 - R4 pooled) at -24, 0 and 24 (fresh media) and at 24 and 48 hours (old media) for quantitative analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

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 (W AF) of the test item.

Definitive test:
At the request of the sponsor, the following loading rates were assigned to the definitive test: 0.20 and 1.0 mg/L. The test was run in duplicate, with one set of samples being centrifuged prior to use and the other set being left to stand in centrifuged tubes and classed as untreated for the purposes of the test.

Amounts of test item (4.5 and 22.5 mg) were each separately added to the surface of 22. 5 liters of test water to give the 0.20 and 1.0 mg/L loading rates respectively. After the addition of the test item, the test water was stirred slowly by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface and slow enough that test item droplets should not disperse into the water column. The media was stirred for 71 hours, with the stirring stopped after 23, 47 and 71 hours and the mixtures allowed to stand for 1 hour prior to removal of samples for use in the test. Nevertheless, 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 (2-4 cm in length). 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 approximate 75-100 mL discarded) to give the 0.20 and 1.0 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that the glass wool plug had removed all of the dispersed test item in the water column.

Once removed, a sample of each WAF was centrifuged (40000 g for 30 minutes). A further sample was placed in identical centrifuge tubes for the period of centrifugation in order to correct for any absorption to the tubes and to investigate if there was undissolved test item present which
would cause effects on the daphnids.

The WAFs removed after 24 hours were used to precondition the test vessels, which were then left to stand for 24 hours under test conditions. After this standing time, the vessels were emptied and refilled with media taken from the 48-Hour preparation period. This was Time-0 of the exposure phase. After a further 24 hours, the vessels were emptied and refilled with the media taken from the 72-Hour preparation period, and all non immobilized daphnids transferred by wide bore pipette into the freshly prepared media. The concentration and stability of the test item in the test preparations were verified by chemical analysis at -24, 0 and 24 (fresh media) and 24 and 48 hours (old media).

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 at approximately 20 °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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Details on test conditions:

In the definitive test 160 mL glass jars containing approximately 160 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 maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light (615 to 720 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

Above water solubility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

Above water solubility

Details on results:

There was no immobilization in 40 daphnids exposed to loading rates of 0.20 and 1.0 mg/L loading rate WAF (untreated and centrifuged) for a period of 48 hours. Inspection of the immobilization data gave the following results:
24 h EL50 (mg/L loading rate WAF) was > 1.0.
48 h EL50 (mg/L loading rate WAF) was > 1.0.
The No Observed Effect Loading rate after 24 and 48 hours exposure was 1.0 mg/L loading rate WAF.

Results with reference substance (positive control):

Analysis of the immobilization data by the trimmed Spearman-Karber method at 24 and 48 hours based on the nominal test concentrations gave the following results:
At 24 h the EC50 was 1.0 mg/L with 95 % confidence limits of 0.91 - 1.2 mg/L. The NOEC was 0.56 mg/L and LOEC 1.0 mg/L.
At 48 h the EC50 was 0.71 mg/L with 95 % confidence limits of 0.65 - 0.76 mg/L. The NOEC was 0.32 mg/L and LOEC 0.56 mg/L.

The No Observed Effect Concentration is based upon zero immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

An estimate of the EL50 values was given by inspection of the immobilisation data.

Chemical analysis of test loading rates

Chemical analysis of the 0.20 and 1.0 mg/L loading rate WAF untreated test preparations in the -24, 0 and 24-Hour fresh preparations showed that measured concentrations of between 0.00117 and 0. 00439 mg/L were obtained, while in the old or expired media at 24 and 48 hours showed that measured concentrations of between less than the LOQ and 0.00853 mg/L were obtained. In some instances, an increase in measured concentration was observed between the old and fresh samples, however, this could be due to the extremely low concentrations that we were seeing and is considered to have no effect on the outcome of the test.

Chemical analysis of the 0.20 and 1.0 mg/L loading rate W AF centrifuged test preparations in the -24, 0 and 24-Hour fresh preparations showed that measured concentrations of between less than the Limit of Quantification (LOQ), determined to be 0. 00021 mg/L and 0. 00305 mg/L were obtained, while analysis of the old or expired media at 24 and 48 hours showed that measured concentrations of between less than the (LOQ), and 0.00195 mg/L were obtained.

The results of the chemical analysis show that overall, lower measured concentrations were observed in the centrifuged samples compared to the untreated samples. This suggests that centrifugation was removing undissolved test item present in the solution which was not visible though microscopic observations of the WAF. This also supports findings of previous solubility work which showed that emulsions found if WAFs of the test item could be removed by centrifugation.

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.

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 ≥3 mg/L in the control and test vessels.

Water quality criteria

Temperature was maintained at approximately 21°C throughout the test, while there were no treatment related

differences for oxygen concentration or pH.

Vortex depth measurement

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 0.20 and 1.0 mg/L loading rates were observed to be a clear

colorless water column with an oily slick of test item on the surface. After each stirring period

and a 1-Hour standing period the 0.20 and 1.0 mg/L loading rates were observed to be clear

colorless water columns with test item on the surface and dispersed throughout. Visual

examination of the WAFs showed that there was test item dispersed throughout the water

column and therefore it was considered justifiable to remove the WAFs by filtering through a

glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass

wool plug had removed all of the dispersed test item whilst leaving the truly dissolved fraction in

solution at the limit of saturation of the test substance. During the test the 0.20 and 1.0 mg/L

loading rates were observed to be clear, colorless solutions.

Cumulative Immobilization Data in the Definitive Test

Nominal Loading Rate (mg/L)	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.20C	0	0	0	0	0	0	0	0	0	0	0	0
1.0 C	0	0	0	0	0	0	0	0	0	0	0	0
0.20U	0	0	0	0	0	0	0	0	0	0	0	0
1.0 U	0	0	0	0	0	0	0	0	0	0	0	0

R₁– R₄= Replicates 1 to 4

C= Centrifuged at 40000gfor 30 minutes

U= Untreated

Water Quality Measurements

Nominal Loading Rate (mg/L)		0 Hours (Fresh Media)			24 Hours (Old Media)			24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C	pH	mg O2/L	T°C	pH	mg O2/L	T°C
ControlC	R1	7.9	7.8	20	7.5	8.9	21	7.7	9.0	22	7.6	8.7	20
Control U	R1	7.9	8.8	21	7.9	8.8	21	7.9	9.0	22	7.6	8.6	20
0.20 C	R1	7.9	7.2	21	8.0	7.6	21	7.9	7.9	22	7.7	7.0	20
0.20 U	R1	7.9	8.4	21	7.9	8.3	21	7.9	8.7	22	7.7	7.4	20
1.0 C	R1	7.9	7.3	21	8.0	7.8	21	7.9	8.1	22	7.6	7.3	20
1.0U	R1	8.0	8.5	21	8.0	8.3	21	7.9	8.7	22	7.7	7.2	20

C= Centrifuged at 40000g for 30 minutes

R₁ = Replicate 1

U= Untreated

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 1.0 mg/L loading rate WAF. The No Observed Effect Loading rate was 1.0 mg/L loading rate WAF. Measured concentrations remained relatively stable over duration of the test, therefore it is considered that the absence of effects was not attributed to absence of the test item.

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).

 At the request of the sponsor, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item at nominal loading rates of 0.20 and 1.0 mg/L (both non centrifuged and centrifuged preparations) for 48 hours at a temperature of approximately 21°C under semi-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 0.20 and 1.0 mg/L loading rate WAF untreated test preparations in the
-24, 0 and 24-Hour fresh preparations showed that measured concentrations of between 0.00117 and 0.00439 mg/L were obtained, while in the old or expired media at 24 and 48 hours showed that measured concentrations of between less than the LOQ and 0.00853 mg/L were obtained.

 

Chemical analysis of the 0.20 and 1.0 mg/L loading rate WAF centrifuged test preparations in the -24, 0 and 24-Hour fresh preparations showed that measured concentrations of between less than the Limit of Quantification (LOQ), determined to be 0.00021 and 0.00305 mg/L were obtained, while analysis of the old or expired media at 24 and 48 hours showed that measured concentrations of between less than the (LOQ), and 0.00195 mg/L were obtained.  (Harlan study number: 41206896). Given the capacity of this substance to form emulsions when high energy preparation methods are used, which may lead to physical effects on the daphnids, every care was taken to expose the animals to solutions of test item that contained either truly dissolved concentrations by centrifugation or stable emulsions by WAF preparation. In both cases, every attempt was made to maintain dissolved concentrations at the highest attainable level by pre absorption of the glassware and by using a semi static method of preparation.

 

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 EL₅₀values of greater than 1.0 mg/L loading rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF.

Description of key information

1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane  has extremely low solubility and a tendency to form dispersions that exhibit physical effects on the test organisms. Recent solubility data has clarified that all older studies were carried out far in excess of the solubility limit without sufficient

separation of undissolved material. Therefore generated data was contradicting and variable and not related to toxic effect. The key

study and the most recent supporting studies have demonstrated that regardless of the nominal loading (1 mg/L or 100 mg/L) of the test material

and providing the appropriate preparation method is used. That the test substance does not cause acute effect to daphnia magna at its solubility limit in test medium.

Additional information

Early ecotoxicity studies on daphnids exposed to the test substance were performed at nominal concentrations and a 48 h EC50 of approximately 40 µg/L (van Wijk & Garttner-Arends, 1998) was determined. Subsequent preliminary studies (Höger, 2010) led to similar conclusions with EC50s around 20 µg/L based on measured concentrations (as detailed in the daphnid acute toxicity robust summary IUCLID submission). Nevertheless, analytical measurements of concentrations of the test substance produced using slow-stir or high energy methods were inconsistent and it was decided to initiate more detailed work on the solubility of the substance.

For this purpose, Mead (2013) prepared two WAFs at 0.1 and 1 mg/L in a set of different test media: deionized water, hydrolysis test medium, daphnid reconstituted medium at two different pHs), at or beyond the expected solubility limit with a stirring vortex gradient of approximately 10%. Further refinements such as filtration and high speed centrifugation of the 1 mg/L WAF were also included. Changes in measured aqueous concentration over time were also considered by analyzing samples at T0 and T400 hours (a suspected potential hydrolytic degradation product, trimethylcyclohexanone, was also quantified to consider a potential loss mechanism of the test substance). From the results of this study, the T0 concentrations achieved using the WAF method (ranging between 20 and 100 µg/L for 1 mg/L WAFs) was found to be far greater than the concentrations in filtered samples (7-14 µg/L) and centrifuged tubes (<2 to 4 µg/L). The 0.1 mg/L WAF concentrations varied between 7 and 80 µg/L). After 400 h of preparation time, the measured concentration of the 1 mg/L WAFs did not change greatly while measured concentrations of the 0.1 mg/L WAFs decreased in the reconstituted media but not in deionized water. The concentration of filtered and centrifuged samples remained similar or decreased slightly in the aged solutions. No increase of the anticipated hydrolytic degradation product was found over time. The substance forms stable emulsions up to about 100 µg/L when prepared in aqueous solutions at concentrations above its solubility limit and the true solubility is that of the centrifuged value (<2 to 4 µg/L) found in this study. A further study (Mullee, 2013) was performed to verify that the substance concentrations found in the centrifugation experiments were not due to loss on the centrifuge tubes and it was confirmed that the substance was in suspension.

For these reasons the early studies by Höger (2011) and that by van Wijk & Garttener-Arends (1999) were performed using high energy techniques causing emulsions to be formed at concentrations greater than the true solubility limit of the registered substance.

Thus all effects value in daphnids found to date were from ecotoxicity studies performed well above the true limit of solubility (as emulsions). Under environmental conditions in which the substance would pass through a WWTP, it would not be expected to be present in the form of an emulsion but would rapidly and strongly adsorb to particulate matter (due to the log Kow >7) and therefore the results from these studies are not considered relevant for risk assessment purposes.

Using a more appropriate test methodology but as a non-GLP screening study, Kean (2013) prepared two series of WAFs for the substance using a slow-stir preparation method at loading rates of 0.2, 0.5, 1.0, 5.0, 10 and 100 mg/L of which one series was centrifuged. At 48 h a maximum of 1 daphnid out of 10 was immobile and this only in WAFs of 5, 10 and 100 mg/L. In the non-centrifuged group no immobilisations were observed at a WAF of 0.2 mg/L, approximately 100 times higher than the determined solubility limit, only 3 out of 10 daphnids were immobilized at 0.5 mg/L and in contradiction with the early studies only 6 out of 10 daphnids were found immobile in the 1 mg/L WAF while only 50% were immobilised at 5 and 10 mg/L. None of the WAFs up to 100 mg/L resulted in 100 % immobilisation (maximum 80% at 100 mg/L).

The most recent and key study by Harris (2013) takes a similar approach to (Kean 2013) in its preparation of the test solutions to ensure adequate removal of undissolved material or dispersions. The measured concentrations observed were considerably closer to the true solubility limit of the test material eliminating the problems observed with earlier studies. No Effects were observed up to

1 mg/L of test material. Furthermore due to the potential for loss of test material in a semi static or static setup and extended flow through sub-chronic supporting study was conducted (Kean 2013) to first brood. This demonstrated that also with continual replacement no acute effects were observed.

The conclusion from the valid studies is that no acute toxicity can be determined at or well above the solubility limit when some care is taken in the preparation of the tests concentrations. No EC50 can be derived from any of the acute ecotoxicity studies and no acute classification is necessary for this substance.