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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-10-18 to 1990-01-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was conducted in accordance with a recognized scientific method for determining acute toxicity to fish. However, during the test a layer of test substance remained visible on the surface of the test media in all test concentrations. Consequently, the LC50 value should be handled with care. with respect to LC50 or other parameters due to the insolubility of the test substance at the concentrations employed.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

The diluting water was a synthetic water, Dutch Standard Water, according to NPR 6507 (1980), having a pH of approximately 8.2 and a hardness of 13°dH, containing per litre of deionized water:
100 mg of NaHC03/1
20 mg of KHCO3/1
200 mg of CaC12.2H20/1, and
180 mg of MgSO4.7H20/l.

The deionized water contained not more than 0.01 mg copper per litre and had a conductivity of less than 5 µS/cm. The TOC-content was not more than 2.0 mg/l.

The selected range of test concentrations was determined in a preliminary range finding test, which was not performed in accordance with GLP. The tested concentrations were: 46 - 100 - 210 - 460 - 1000 mg/1. They were prepared by direct addition of the test substance to the diluting water, because the test substance did not dissolve in acetone either. For results of the range finding test see Table in "Any other information on materials and methods inc. tables".
In the definitive test the test concentrations were prepared by direct addition of the respective amounts of test substance to the diluting water.

To reach an equilibrium the test media were stirred for 24 hours at room temperature. After this time the fish were introduced (10 fish per test concentration and control).
During the test the test substance floated as a layer on the water surface.

Test organisms (species):

Pimephales promelas

Details on test organisms:

In contravention to the study plan they were obtained from the RIZA (Rijksinstituut Zuivering Afvalwater). The fish were held in stock for 13 days in the Akzo Research Laboratories Arnhem, dept. CRL, in conformity with Standard Operating Procedures SOP CRL T9 and T42 to determine the acceptability of the batch. The size of the fish was approximately 2 cm.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period was realised.

Hardness:

The diluting water was a synthetic water, Dutch Standard Water, according to NPR 6507 (1980), having a hardness of 13°dH, containing per litre of deionized water:
100 mg of NaHC03/1
20 mg of KHCO3/1
200 mg of CaC12.2H20/1, and
180 mg of MgSO4.7H20/l.

Test temperature:

In contravention to the study plan the temperature was kept between 24° and 25°C and the light regime was 12 hours of ambient light per day.

pH:

pH of approximately 8.2
pH measurements on days 0, 2 and 4. If a high mortality was observed on the other days, the oxygen concentration and pH were measured then also.

Dissolved oxygen:

Measurements of the oxygen concentration were carried out on days 2 and 4. If a high mortality was observed on the other days, the oxygen concentration and pH were measured then also.

Salinity:

Test was performed in freshwater.

Nominal and measured concentrations:

The selected range of test concentrations was determined in a preliminary range finding test, which was not performed in accordance with GLP. The tested concentrations were: 46 - 100 - 210 - 460 - 1000 mg/1. They were prepared by direct addition of the test substance to the diluting water, because the test substance did not dissolve in acetone either. For results of the range finding test see Table in "Any other information on materials and methods inc. tables".
In the definitive test the test concentrations were prepared by direct addition of the respective amounts of test substance to the diluting water.

Details on test conditions:

The fish are exposed to various concentrations of the test substance in diluting water for 96 hours in a semi-static test. Mortalities are recorded at 24, 48, 72 and 96 hours and the concentrations which kill 50 % of the fish (LC50) are determined at 24, 48, 72 and 96 hours where possible.
Effects may be caused either by exposure to the concentrations of test substance dissolved in the water or via contact with or ingestion of particles of the test substance. Furthermore, the highest concentration tested causing no mortality (NOEC, no observed effect concentration) and the lowest concentration tested producing 100 % mortality within the period of the test are recorded where possible.

As test vessels were used 2-1 covered glass aquaria which contained 1 litre of test medium.
The loading of the test vessels was approximately 0.6 g biomass/1.
The surviving fish were counted daily and dead fish removed immediately upon observation.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

560 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

750 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The LC50 {96 h) as calculated was 750 mg/1 with 95% confidence limits of 360 and 15-70 mg/1. However, given that during the test in all test concentrations a layer of test substance remained visible at the surface of the test media, the LC50 value should be handled with care.
At all test concentrations the fish developed serious deviations in behaviour and appearance during the test period, such as reduced activity, convulsive movements and floating on the back. Most of the fish developed a thick white stomach.

After 96 hours no mortality occurred at test concentrations containing up to and including 560 mg/1 while 100% mortality occurred after 96 hours at 1000 mg/1 and after 24 hours at 1800 mg/1.

See table in "Any other information on results incl. tables".

Results with reference substance (positive control):

No data.

Reported statistics and error estimates:

No data.

Sublethal observations / clinical signs:

Mortality in definitive test

		number of surviving animals
mg test substance/1	0	24	48 hours	72	96
control I	10	10	10	10	10
control II	10	10	10	10	10
320	10	10	10	10*	10*
560	10	10*	10*	10*	10*
1000	10	9*	8*	5*	0
1800	10	0
temperature	24	25	24	25	24
oC

* serious deviations in behaviour and appearancesuch as reduced activity, floating on the back, thick white stomach, convulsive movements.

Statistical evaluation

time (d)	LC      (g/1)	95% confidence limits
1.0	1.09	1.01 - 1.18
2.0	1.05	0.99 -1.12
3.0	1.00	0.95 - 1.05
4.0	0.75	0.36 -1.57

Validity criteria fulfilled:

yes

Remarks:

See conclusions. Study acceptable with restrictions.

Conclusions:

The quality criteria of this test have been fulfilled: the mortality in the control was 510%, the oxygen concentration was >60% of the air saturation value throughout the test (in earlier tests the air saturation value was determined to be approximately 8.5 mg 02/1). In view of the results of a biodegradability study (C.G. van Ginkel, 1990. Biodegradability of [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide, it is likely that the concentration of the test substance remained constant during the test.
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was tested in an acute toxicity test with Poecilia reticulata (guppy) under semi-static conditions in accordance with EEC method C.1. (1984) and OECD Test Guideline 203 (1984).
At all concentrations tested the fish developed serious deviations in behaviour and appearance during the test period, such as reduced activity, convulsive movements and floating on the back. Most of the fish developed a thick white stomach.
After 96 hours, no mortality was observed up to and including 560 mg/1 and 100% mortality was observed at 1000 mg/1 after 96 hours and at 1800 mg/1 after 24 hours.
The LC50 (96 h) was calculated to be 750 mg/1, with 95% confidence limits of 360 and 1570 mg/1.
However, given that during the test a layer of test substance remained visible on the surface of the test media in all test concentrations, the LC50 value should be handled with care. Moreover no analytical monitoring was performed during the test.
Based upon the fact that no mortality was observed this test can be considered as valid with restrictions.

Executive summary:

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was tested in an acute toxicity test with Poecilia reticulata (guppy) under semi-static conditions in accordance with EEC method C.1. (1984) and OECD Test Guideline 203 (1984).

At all concentrations tested the fish developed serious deviations in behaviour and appearance during the test period, such as reduced activity, convulsive movements and floating on the back. Most of the fish developed a thick white stomach.

After 96 hours, no mortality was observed up to and including 560 mg/1and 100% mortality was observed at 1000 mg/1 after 96 hours and at1800 mg/1 after 24 hours.

The LC_s0(96 h) was calculated to be 750 mg/1, with 95% confidencelimits of 360 and 1570 mg/1.

However, given that during the test a layer of test substance remained visible on the surface of the test media in all test concentrations, the LC₅₀value should be handled with care. Moreover no analytical monitoring was performed during the test. Based upon the fact that no mortality was observed this test can be considered as valid with restrictions.

Reference 2

Endpoint:

fish embryo acute toxicity (FET)

Remarks:

Combined report in which existing fish data gaps were filled with FET screening data for multiple substances

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Collection of OECD236 data for various substances

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Version / remarks:

Test conducted in a screening form with limited concentrations and no chemical analysis.

Deviations:

yes

Remarks:

Not GLP, No Chemical analysis,

Principles of method if other than guideline:

Screening method used a higher number of Embryos are used per well. Daily refreshment from a water accommodated fraction containing and excess of the parent material. When the parent material is visibly in excess and stirred for an extended period the water fraction can be considered saturated with the parent material and any accumulating degradation products or impurities at their corresponding solubility limits. Test material is of extremely low water solubility lower than the detection limit of available analytical methods. Hence daily refreshment was utilized to maximise exposure.

GLP compliance:

no

Remarks:

Conducted to simplified version of the guideline OECD 236 study

Specific details on test material used for the study:

batch no.: 1403454131

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

no

Details on test solutions:

Test solutions
When the solubility and stability of the material was known to be low WAF (Water accommodated fraction) solutions were made to prepare the test material.

Slow stir WAF preparations have been shown to be capable of producing a stable concentration of dissolved parent material (as well as stabilizing agent or accumulating degradation products) when loaded in excess of the water solubility and slowly stirred in the same manner as a slow stir water solubility test.

Each WAF vessel was equipped with a Teflon tap that allows only the water accommodated fraction to be transferred without the transfer of undissolved material.

The test material for the WAF prepared substances was weighed accurately and added to each WAF vessel separately. The vessels were then carefully filled with 1 liter of test medium and stirred slowly under sealed conditions at room temperature for approximately 72 hours. Prior to the test the stirring was stopped and all WAF vessels were left to rest for 1 hour after which all of the test wells were rinsed with the appropriate test solution to minimize the absorbance of the test material. The test solutions were then discarded and refilled prior to testing. WAF vessels were then restarted and used for the daily refreshment of the solutions in the same manner.

Solution replenishment
Solution refreshment was carried out daily for WAF preparations by removing as much liquid as possible from each well (while avoiding drying of the embryos) using a pipette. After which the appropriate WAF solution was tapped off directly into the test wells. The WAF solutions (in which excess of test material remained visible) were used at the start of the test as well as for all solution replacements.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Embryos arrived 3-4 hpf (hours post fertilization). As soon as the test solutions were made embryos were divided into bulk batches using glass pipettes at the appropriate concentration for each test chemical to prevent delay in exposure caused by preparation time. The wells were then filled with each of the stocks made for each chemical at each concentration. After a maximum of one hour the wells were emptied and refilled, after which the embryos (5 per well) were added. Plates were then incubated for the test duration and observed daily.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Daily replenishment of test solutions from a WAF containing an excess of test material.

Limit test:

no

Total exposure duration:

96 h

Hardness:

The test medium Dutch Standard Water (DSW) was used for testing. DSW has a , conductivity of 550-650 µS/cm, and contains: 200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 per liter. The water was made by addition of the stocks and continual aeration before being used in the test.

Test temperature:

26ºC +/- 2ºC

pH:

pH of 8.2

Dissolved oxygen:

Continually aerated before use

Salinity:

N/A

Conductivity:

550-650 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 1,10 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Wellplate
- Type: closed
- Material, size, headspace, fill volume: 3mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

OTHER TEST CONDITIONS
- Adjustment of pH: Not required

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: screening (fish data not compulsory at registered tonnage)

Reference substance (positive control):

yes

Remarks:

Internal positive and negative controls acceptable

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Extended stir parent + Degraded material

Basis for effect:

coagulation of the embryo

Details on results:

Test material can be concluded to have an LC50 / EL50 in excess of its water solubility limit for fish embryos / hatchlings.

Results with reference substance (positive control):

Acceptable according to guideline

Reported statistics and error estimates:

Range estimated in comparison to control only

Validity criteria fulfilled:

yes

Remarks:

With restrictions Not GLP and No Chemical analysis. Valid therefore as supporting evidence.

Conclusions:

An estimation of the EL50 for adult fish species was made for the material tested. The validity criteria for positive and negative controls were met and where this was not the case this was justified accordingly. The embryo batches were considered of good quality and sufficient for use in the OECD 236 test. The test material was not toxic to the test embryos or hatchlings at the water solubility limit of the test substance.

Executive summary:

Guideline study in simplified screening form without GLP and without chemical analysis. Required validity and reference criteria were met. Data considered an acceptable nominal estimation for the marketed product. Daily refreshment and well rinsing used to maximise exposure.

Description of key information

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was tested in an acute toxicity test with Poecilia reticulata (guppy) under  semi-static conditions in accordance with EEC method C.1. (1984) and OECD Test Guideline 203 (1984). The LC50(96 h) was calculated to be 750 mg/L, with 95% confidencelimits of 360 and 1570 mg/L.

This is confirmed by a non-GLP FET according to OECD 236: Test material can be concluded to have an LC50 / EL50 in excess of its water solubility limit for fish embryos / hatchlings.

Additional information

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide was tested in an acute toxicity test with Poecilia reticulata (guppy) under semi-static conditions in accordance with EEC method C.1. (1984) and OECD Test Guideline 203 (1984). At all concentrations tested the fish developed serious deviations in behaviour and appearance during the test period, such as reduced activity, convulsive movements and floating on the back. Most of thefish developed a thick white stomach. After 96 hours, no mortality was observed up to and including 560 mg/L and 100% mortality was observed at 1000 mg/L after 96 hours and at1800 mg/L after 24 hours. The LC50(96 h) was calculated to be 750 mg/L, with 95% confidence limits of 360 and 1570 mg/L.

However,given that during the test a layer of test substance remained visible on the surface of the test media in all test concentrations,the LC50 value should be handled with care. Moreover no analytical monitoring was performed during the test. Based upon the fact that no mortality was observed this test can be considered as valid with restrictions.

The test results are confirmed by a non-GLP Fish Embryo test according to OUECD Guideline 236. Nominal test concentrations of 1, 10 and 100 mg/L did not show significant effects and therefore it can be concluded that the test substance has an EL50 > 100 mg/L or greater than the limit of water solubility.