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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.09.2013 - 4.11.2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study to OECD guideline with justified modifications for test material properties. Restricted by the quantification limit of the analytical method.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Adaptions for poorly soluble, adsorbing, reactive test materials

Principles of method if other than guideline:

1)The test substance has very low measured water solubility (approximately 2 µg/L). Difficulties have occurred in obtaining reproducible solubility values due to the tendency of the test substance to adsorb strongly and disperse easily. Likewise variable acute toxicity data has been generated due to some studies unintentionally being conducted above the water solubility of the test substance and the propensity for the substance to form temporarily stable emulsions when using high energy techniques for stock generation. For these reasons and due to the available guidance recommending that the water solubility not be exceeded, a customized flow through setup was built to enable testing with a more continual exposure at the maximum obtainable concentration using a low energy slow stir method. The intention being that the exposure is closer to the true solubility of the test substance.

2)Preliminary (flow through) studies have shown difficulties in maintaining a steady exposure in the presence of increasing levels of organic material inherent to a chronic daphnia test. For this reason the system was primed 48 hours prior to addition of test animals to saturate the test apparatus.
Regular cleaning of all test apparatus was carried out throughout the test period in order to reduce the level of organic material in the test system thereby reducing adsorption as much as possible.

3)To ensure that analytical determinations could additional analytical measurements of test solutions in the aquaria, at the inlets and in the stock (WAF) were made. The extremely low solubility of the test substance meant that analytical measurement was not always possible.

5)Organic peroxides have the tendency to react with metal ions, reducing the concentration of biologically available iron for the test organisms. The potential effects are therefore not related to toxicity but to essential element bioavailability. The level of iron in the test medium was therefore be elevated to (125% of the recommended concentration) in order to compensate for the removal of iron by the test substance.

6)Due to potential interaction of the test substance with algae cells used for feeding contact time with algae was minimized as far as possible. Algae was fed three times a day during work days directly into the test aquaria. In the weekends this was achieved by a pumping system adding algae to the intermediate overflow aquarium. (See apparatus description on page).

7)The most recent OECD 211 guideline requires the reproduction endpoint to be expressed as total number of offspring per surviving adult at the end of the test as well as for the total number of adults at the start of the test. Due to the flow-through design employed in this test, where aquaria contain replicates of 10 organisms the use of all statistical tests proposed is not possible. Total reproduction per adult at the start of the test only will therefore be used for expression of the reproduction endpoint.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Sampling took place on Day 0,2,6,13,16,19,20 and 21 in the WAF the overflow aquarium all inlets and all tanks containing test substance and a single sample of test medium from the control. On day 20 and 21 all tanks were additionally sampled also for the control to give a more accurate indication of the background caused by organic material.

6 ml samples were taken and were added to 4 ml acetonitrile and vortexed prior to analysis. In the event that samples were stored appropriate stability samples were made to demonstrate storage stability.

Vehicle:

no

Details on test solutions:

WAF (Stock Solution)
The test material was loaded at a nominal concentration of 15 mg/L to 30L aquaria containing modified M4 medium. Slow stirring and aeration was
started as long as possible prior to use of the stock to allow equilibration of the test material. Essentially the stock solution was a large water accommodated fraction (WAF) used to continually feed the test replicates. The test substance stock or WAF was renewed at least three times per week to ensure continual exposure despite the tendency of the stocks to decrease in concentration due to adsorption of the test material. The stock solution was
pumped continually via the setup described in the details on test conditions section..


Test medium
The test medium was prepared by mixing de-ionised water with standard Elendt M4 components as detailed in the OECD 211 study guideline, with the following modifications: The Fe-EDTA concentration was intentionally elevated to 125% of the recommended guideline concentration.
The concentrations of MnCl2.4H20, LiCl, RbCl ,SrCl2.6H20, CuCl2.2H2O, ZnCl2 and CoCl2.6H20 were decreased to 25% the recommended
concentration.

The dilution water was aerated continually by the test setup. The air used was water-saturated and purified by activated charcoal and a cotton filter.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test animals were taken from a Daphnia magna clone 5 stock, (origin: Wil Research (Formerly Notox), The Netherlands). The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenetically and aged between 2-4 weeks having previously produced at least one brood before use. Offspring from the Daphnia culture were reference tested with potassium dichromate twice annually in
the relevant dilution water. The culturing of the test animals was in accordance with laboratory Standard Operating Procedures.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Animals were inspected post exposure for physical appearance and were measured for the length endpoint only. No post exposure observation
period as such.

Hardness:

280 mg/L as CaCO3.

Test temperature:

20.3-21.4ºC

pH:

pH measurements did not fall below 7.9 and did not exceed 8.2.

Dissolved oxygen:

The measured oxygen concentration did not fall exceed 9.5 mgO2/L.

Salinity:

N/A

Nominal and measured concentrations:

15 mg/L Nominal. 12.8µg/L ( average measured) [pumped directly into test vessels]. <4 µg/L [test vessels].

Details on test conditions:

Description of experimental setup
An accurately weighed amount of approximately 0.45 g the test substance was loaded to glass (stock) 30L aquaria filled with test medium. The control groups were fed from a single 80L aquarium. The (stock) aquarium was mechanically agitated with a magnetic stirrer at the lowest possible speed using large Teflon® coated stirring bars. The control group medium was not agitated. All aquaria were gently aerated.

The test solution was pumped continually from stock aquaria with a single multi head peristaltic pump via a glass frit and glass mixing column to a small glass overflow aquarium (5L) intended to further separate the test material and act as a receptacle for algae during the weekend period. Liquid was either recycled back to the stock aquaria or to drain. Test replicates were then fed by a second identical pump with multiple tubes made from chemically inert, plasticiser free tubing, extracting liquid from the middle of the overflow aquarium directly feeding the 3 test replicates.

The control replicates were fed in an identical manner by the same pumps using identical tube types and diameters but directly from an 80L aquarium containing test medium only. A mixing column and glass frit were not used for the control. In addition two 30L aquaria were used for the test substance to allow stock preparation in advance so as to allow maximum stabilization of the soluble component of the stock solution and enough volume to bridge the weekend periods.

Apparatus (Flow through setup)
Both the control and the test concentration will be pumped at the desired flow rate using calibrated Gilson peristaltic pumps equipped with Viton® tubing. Fluid will be transferred through Norprene® tubing or glass. The chosen tubing was selected due to its strong chemical resistance, an inner bore
that is resistant to adsorption/absorption of aqueous fluids as well as a lack of plasticisers (in the bore).

Silicone and PVC tubing was not be used (except for drain tubes) as this was shown to degrade in preliminary studies.

Renewal Rate
The test replicates and control aquaria were renewed automatically by pumps as previously described. At least 5 x the volume of the aquaria (5 x 400 ml) each day was pumped into each replicate. This was determined by the pump calibration before the start of the test. Pump calibration speed was based on the tube with the lowest delivery. The calibration was checked once during the test and pumps adjusted accordingly.

Test Vessels
Glass aquaria with an approximate volume of (400 ml at outlet) were used as test vessels in triplicate for the control and in triplicate for the only test
concentration. The test vessel was continually fed via a glass inlet pipe on the opposite side of the outlet. 10 test animals per aquarium were used for
testing.

Reference substance (positive control):

yes

Remarks:

Conducted as part of routine laboratory maintenance

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: No significant difference to control detected

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

12.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

Measured in secondary separation tank directly feeding all test substance replicates. Generated by a nominal loading of 15 mg/L (above)

Basis for effect:

reproduction

Remarks on result:

other: No significant difference to control detected

Remarks:

Limited by presence of algae

Details on results:

The test results demonstrate that test replicates fed with an average concentration of 12.8 µg/L resulted in no detectable reproduction effects.

Results with reference substance (positive control):

The daphnia culture is periodically checked for sensitivity as part of the routine GLP laboratory maintaince using potassium dichromate. The most
recent reference test resulted in an EC50 of 0.7 mg/L, which is acceptable according to the EEC ring test criteria.

Reported statistics and error estimates:

Data was evaluated using a two sample statistical method Homoscedastic t-Test. Intended for comparison of a single treatment with a single control.

Results.

Control

Day of Study	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of juveniles																						Total
1 (Number of adults)	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	9	9	9	9	7 1￥	7	7	2M 1￥
2 (Number of adults)	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	9	9	9	1M
3 (Number of adults)	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	9	9	9	7	7	7	3M
Number of Juveniles 1 [N]	-	-	-	-	-	-	-	√	33	18	-	√	249	168	√	188	54	-	-	88		301	1099
Number of Juveniles 2 [N]	-	-	-	-	-	-	-	√	27	59	-	√	197	133	√	195	68	-	-	112		337	1128
Number of Juveniles 3 [N]	-	-	-	-	-	-	-	-	52	40	-	√	301 3*	66	√	293	27	-	-	87		253	1119
Total mortality of parent groups [%]																							76 (80)￥

15 mg/L

Day of Study	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of juveniles																						Total
1 (Number of adults)	10	10	10	10	10	10	10	10	10	10		10	10							9	9	9	1M
2 (Number of adults)	10	10	10	10	10	10	10	10	10	10		9	9	9	8	8	8			7	7	7	3M
3 (Number of adults)	10	10	10	10	10	10	10	10	10	10		10	10	10	10	9	8			8	8	8	2M
Number of Juveniles 1 [N]	-	-	-	-	-	-	-	√	55 3*	54		√	272		33 16AE	353	93			113		328	1301
Number of Juveniles 2 [N]	-	-	-	-	-	-	-	-	39 4*	47		√	305		198	146	93			123		240	1191
Number of Juveniles 3 [N]	-	-	-	-	-	-	-	√	49	48		√	175 2*		227	167	203			145		206	1220
Total mortality of parent groups [%]																							80

– = no juveniles

￥=Inadvertant mortality

* = immobile / aborted juveniles

√ = First Brood/Juveniles present

M = Mortality

E= Aborted eggs

Analytical Results

Sample	Day of sampling							Average
	D0	D2	D6	D13	D16	D20	D21
Control Tank I	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Control Tank II	n.d.	n.d.	n.d.	n.d.	n.d.	< LOQ	< LOQ
Control Tank III	n.d.	n.d.	n.d.	n.d.	n.d.	< LOQ	< LOQ
Control Inlet I	n.d.	n.d.	n.d.	n.d.	n.d.	< LOQ	n.d.
Tank I	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Tank II	8.7	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Tank III	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
Inlet I	< LOQ	< LOQ	8.2	< LOQ	< LOQ	< LOQ	< LOQ
Inlet II	9.2	< LOQ	8.0	< LOQ	< LOQ	< LOQ	< LOQ
Inlet III	< LOQ	< LOQ	9.1	< LOQ	< LOQ	< LOQ	< LOQ
WAF overflow	16.5	11.8	18.8	9.7	7.2	< LOQ	< LOQ	12.8
WAF	17.7	99.5*	53.6	13.6	8.2	23.8	21.1	33.9

Analytical Quality Criteria

Parameter	Limit	Determined
Correlationcoefficient (R^2)	≥ 0.98	≥ 0.992
Accuracy (%)	80 - 120	83.3 – 118.7
Quality Control (QC) samples (%)	80 - 120	91.6– 106.5
Repeatability (CV (%)) at lowest standard	≤ 20	≤ 16.1
Repeatability (CV (%)) at Highest standard	≤ 20	≤ 2.5
LOQ (µg/L)	10	4.0
System stability (% of nominal)	≤ 10	≤ 8.3
Recovery stability samples (%)	≤ 20	5.8

Validity criteria fulfilled:

yes

Conclusions:

In conclusion the test material caused no detectable chronic effects to the test organisms at a nominal loading of 15 mg/L that after several
separation steps resulted in an exposure concentration at the solubility limit of the test material. Due to the continual dosing the result can be
considered representative of an absolute worst case scenario. In reality the test material is unlikely to pass through water treatment processes due to its tendency to adsorb strongly to organic matter.

Executive summary:

A GLP study to appropriate guideline with justified modifications considering test substance properties. Certificate of analysis and chemical analysis were present. A modified test setup to avoid high energy dispersions above solubility limit was used. Technical limits restricted the quantification limit of the analytical method to 4 µg/L. This prevented measurement (for the vast majority of vest vessel samples) in the test replicates themselves. This was due to the 2 µg/L solubility limit of the test material. Analysis did however demonstrate continual presence of the test material at an average concentration of 33.9µg/L in the stock (WAF) solution that was continually pumped into the test system. Furthermore the secondary separation vessel (directly feeding the test vessels) maintained an average concentration of 12.8 µg/L throughout the test period. This is considered the best attempt possible at this time due to technical limitations of the analytical apparatus used. Due to further separation taking place after the secondary separation vessel and potential reaction with organic material in the test vessels detection in the test vessels themselves was not possible in the majority of samples taken. However during the priming of the test system and occasionally throughout the test the inlet and tank measurements the test material was detected above the LOQ. It is therefore fair to assume that considering the continual dosing and relitively high nominal loading of 15 mg/L, that the best possible exposure took place in the test vessels. The study is not without limitations however.

Description of key information

No effects on the reproduction of Daphnia Magna were observed in a chronic flow through study at the limit of solubility of the test substance in the

test medium.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

12.8 µg/L

Additional information

A flow through study with a nominal loading of 15 mg/L of the test substance was conducted using a slow stir method (WAF) and two stage separation of the stock solution to avoid the testing of dispersed material. The stock (or large WAF solution 30L) was continually pumped into the test system the WAF itself was refreshed 3 times per week to ensure continual presence of the test material. Analytical measurements demonstrated the presence of the test material in the WAF solution throughout the test as well as in the secondary separation vessel at concentrations above the limit of solubility. From the secondary separation vessels triplicate test vessels containing 10 test organisms were exposed to the test substance for 21 days over which primarily the effects on reproduction were assessed.

No siginificant effects on reproduction or body length were observed. Indicating that 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane causes no chronic effects to Daphnia magna at it's water solubility limit. No chronic classification is required for this substance based on this data.