Di-tert-pentyl peroxide

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:

June 2019 - July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Deviations were considered to have not affected the outcome or the achievement of the study objectives. See "Any other information on results incl. tables"

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control on fresh and old solutions for each occasion. The test item concentrations were determined on the same day as sampling under the analytical procedure

Vehicle:

no

Details on test solutions:

See "any other information on materials and methods incl. tables".

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organisms used for this study was Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by a cyclical parthenogenesis in a synthetic medium. D. magna cultures were fed with a mixture of the microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny. At the start of the experiment one D. magna neonate was added to each vessel.

Test animals were fed a diet of around 0.1 - 0.2 mg of carbon per daphnid per day, in the form of a suspension of the algal strains Chlorella vulgaris (25%) and Pseudokirchneriella subcapitata (75%), except during the initial three to five days of culture when a slightly lower ration was given. The concentrated algal cell suspension was prepared by removing and centrifuging aliquots of algal culture and resuspending the algal pellets in small volumes of Volvic® water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The total hardness (as CaCo3) : 170 mg/L

Test temperature:

Temperature was measured as follows: min. 19.0°C, max. 20.2°C and mean: 19.6°C.

pH:

pH: 7.4-8.5

Dissolved oxygen:

dissolved oxygen concentration: 8.5-10.0 mg/L.

Nominal and measured concentrations:

No preliminary test
Definitive test concentrations: series of dilutions 7.0, 12.0, 20.4, 34.6, 58.8 and 100.0% equivalent to mean measured concentrations : 0.6, 1.2, 2.0, 3.3, 5.2, 13 mg/L

Details on test conditions:

Test vessels containing D. magna were incubated at a temperature ranging from 18°C to 22°C, the temperature did not vary by more than 2°C as daily range. A light cycle of 16 h light and 8 h dark was in operation throughout the test, provided by LED light (light intensity around 800 lux).
Test vessels were made of clear glass tubes (around 180 mL capacity) and stoppered with cellulose bungs to minimise evaporation and avoid the entry of dust into the solutions. At least 50 mL of exposure solution was provided for each organism.
The control treatment was maintained under the same conditions as the treated groups, except it did not contain any test item. No auxiliary substance was used.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

See "Any other information on results incl. tables"

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated monthly by performing an acute Daphnia magna toxicity test on potassium dichromate. The nearest value of 24h-EC50 obtained on May 2019 was 1.0 mg/L.
The ISO 6341 norm has a validity criterion specifying that the reference item EC50 must be in the 0.6 to 2.1 mg/L range.

Definitive Reproduction Test

All test solutions appeared clear and colourless. No precipitation of test item nor undissolved particles were observed.

The analytical results data are presented in Table below. Chemical analysis of test samples confirmed the unstability of the substance over a 24-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

Concentrations used for statistical analysis

Test solution	Test item measured concentrations(mg/L)																		Exposure conc. (mg/L)*
	Occasion 1			Occasion 2			Occasion 3			Occasion 4			Occasion 5			Occasion 6
	fresh	old	% dev. 24h	fresh	old	% dev. 24h	fresh	old	% dev. 24h	fresh	old	% dev. 24h	fresh	old	% dev. 24h	fresh	old	% dev. 24h
Control	<DL	<DL	NA	<QL	<QL	NA	<DL	<DL	NA	<DL	<DL	NA	<DL	<DL	NA	<DL	<DL	NA	NA
7.0	0.9	0.24	-73.3	1.3	0.36	-72.3	0.86	0.37	-57.0	0.99	0.26	-73.7	1.43	0.39	-72.7	1.51	0.44	-70.9	0.6
12.0	2	0.62	-69.0	2.3	0.61	-73.5	1.94	0.65	-66.5	1.79	0.57	-68.2	2.42	0.67	-72.3	2.44	0.73	-70.1	1.2
20.4	3.39	1.04	-69.3	3.6	1.06	-70.6	3.45	1.21	-64.9	3.23	0.96	-70.3	4.06	1.24	-69.5	3.83	1.25	-67.4	2.0
34.6	5.43	1.46	-73.1	5.92	1.81	-69.4	6.53	2.08	-68.1	5.39	1.69	-68.6	6.32	1.95	-69.1	6.33	1.99	-68.6	3.3
58.8	8.45	2.94	-65.2	9.35	3.03	-67.6	8.94	3.71	-58.5	6.42	2.75	-57.2	9.45	3.31	-65.0	9.05	3.43	-62.1	5.2
100.0	14.9	ND	NA	16.2	ND	NA	16.2	ND	NA	11.3	ND	NA	12.8	10.2	-20.3	16.8	8.18	-51.3	13.0

ND: not determined NA: not applicable

< DL: concentration lower than the Detection Limit (0.007 mg/L)

< QL: concentration lower than the Quantification Limit (0.022 mg/L)

*concentrations expressed as geometric means of measured concentration

The following deviations were observed in this study:

- The analysis conducted the first week of the test have shown unexpected values and thus new analysis were conducted with another analytical technique (UPLC-MS-MS rather than GC-MS-MS). The results obtained were compliant with the one expected considering the test item saturation in water (i.e. 13.8 mg/L).

- The analytical results at the test item solution of 100.0% for old solution are missing for occasion #1 to #4: as all daphnids died the first week of the test, the test solution 100% was not kept in the incubator to get the corresponding old solution. To fix this issue 2 additional occasions #5 and # 6 were added during which one test solution at 100% was aged 24h within exactly similar test conditions except that no daphnia was introduced in the test vessel.

These deviations were considered to have not affected the outcome or the achievement of the study objectives.

A summary of all biological data is presented in the table hereafter:

	Test solution (% saturation)
	Control	7.0	12.0	20.4	34.6	58.8	100.0
Total of living offspring (per parent alive)*	758	812	816	560	296	0	0
Mean of living offspring produced per parent	75.8	81.2	81.6	56.0	32.9	0.0	n.a.
Standard Deviation	15.2	25.2	37.7	13.1	21.3	0.0	n.a.
Total of dead offspring	7	5	6	24	32	30	0
Total of egg	4	0	1	2	2	1	0
Total of dead parent	0	0	0	0	1	2	10

*produced at the end of the test by all the parent daphnia integrated at the start of the test excluding parental accidental and/or inadvertent mortality

n.a.: not applicable, all organisms died

Mobility inhibition

The number of immobile adult D. magna recorded during the test were 1 (10%) at 34.6%, 2 (20%) at 28.8% and 10 (100%) at 100.0. No immobile D. magna were recorded at others test item concentration or in the control. The no observed effect concentration (NOEC) was determined to be 3.3 mg/L (expressed as geometric mean of measured concentrations).

Reproduction inhibition

A statistical analysis of complete data shown significant effect on Daphnia reproduction at the test solution 20.4% (i.e. 2.0 mg/L expressed as geometric mean of measured concentrations).Thus NOEC = 1.2 mg/L (expressed as geometric mean of measured concentrations).

Age at First Reproduction

Offsprings were observed in the 7.0, 12.0, 34.6% treated group by Day 8, by day 9 in the control and 20.4% groups, and by day 12 in the 58.8% group. No offspring were observed in the 100% treated group. This difference was statistically considered as significant: NOEC = 2.0 mg/L (expressed as geometric mean of measured concentrations).

NOEC, LOEC, EC10 and EC50 values on day 21 were calculated to be as follows:

Endpoint: Mean number of neonates produced per survivor		95% confidence level
		Lower limit	Upper limit
21d-EC10	1.6 mg/L	1.2	2.1
21d-EC50	2.8 mg/L	1.9	3.9
NOEC	1.2 mg/L
LOEC	2.0 mg/L

Endpoint: Parent mobility		95% confidence level
		Lower limit	Upper limit
21d-EC10	3.9 mg/L	2.3	4.9
21d-EC50	6.3 mg/L	4.9	9.2
NOEC	3.3 mg/L
LOEC	5.2 mg/L

Endpoint:Age at first brood		Endpoint:Age at first brood
		Lower limit	Upper limit
21d-EC10	nd	nd	nd
21d-EC50	nd	nd	nd
NOEC	2.0 mg/L
LOEC	3.3 mg/L

nd: not determined for statistical reasons

Environmental conditions:

Test solution quality parameters (pH and dissolved oxygen concentration) were measured in one replicate vessel at each concentration. All parameters remained within the protocol specifications and remained within the following ranges: pH: 7.4-8.5 and dissolved oxygen concentration: 8.5-10.0 mg/L.

Temperature was measured as follows: min. 19.0°C, max. 20.2°C and mean: 19.6°C.

The light intensity was measured between 600 and 1051 lux

The total hardness (as CaCo3) of the test medium used to prepare test solutions was determined on fresh solutions using a Hardness test kit.

Validation Criteria

The definitive test met the validity criteria of the test guideline detailed as follows (for the control group):

- the mortality of the parent animals (female Daphnia) did not exceed 20% at the end of the test (0%);

- the mean number of living neonates produced per parent animal surviving at the end of the test is > 60 (75.8)

In addition the CV (20.1%) of the mean number of living neonates produced per control parent animal surviving at the end of the test was lower than the threshold recommended by the OECD 211 guideline (25%).

Validity criteria fulfilled:

yes

Conclusions:

In this study, the lowest NOEC (1.2 mg/L) and the 21d-EC10 (1.6 mg/L) was observed for the mean number of neonates produced per surviving parent.

Executive summary:

This study was designed to determine the effects of Di-tert-amyl peroxide on Daphnia magna reproduction and survival in a 21 days test according to the OECD 211 Guideline (2012).

Daphnia magna were exposed to a series of dilutions (7.0, 12.0, 20.4, 34.6, 58.8 and 100.0%) of a saturated stock solution, together with a control group. The test solutions (including control) were renewed daily throughout the test, including weekends. The test solutions were prepared by gently adding the test item at the surface of the test medium to obtain a loading rate of around 50 mg/L. The solution was stirred during approximately 24 hours then drawn off as a clear solution. This saturated solution was then further diluted to obtain each test solution. Ten replicate test vessels were prepared for the control and for each test concentration. A single juvenile Daphnia magna (<24 hours old selected by filtration) was added to each test vessel. On renewal occasion, parental animals were transferred into freshly prepared test medium. Any juveniles remaining in old test media were counted and the number of juveniles produced per parental animal on any day during the 21-day study duration was recorded. The time of the first production of young, the number of young born, immobility and other signs of toxicity observed were compared to a control group.

Chemical analysis was made on fresh and old solutions throughout the test. A decline in measured test concentration of the aged test solutions was observed. Based on these results, the exposure concentrations were based on the geometric means of measured concentrations. NOEC and LOEC after 21 days of exposure were calculated to be as follows:

Endpoint	NOEC	LOEC
Mean number of neonates produced per survivor	1.2 mg/L	2.0 mg/L
Parent mobility	3.3 mg/L	5.2 mg/L
Age at first brood	2.0 mg/L	3.3 mg/L

In this study, the lowest NOEC (1.2 mg/L) and the 21d-EC10 (1.6 mg/L) was observed for the mean number of neonates produced per surviving parent.

The definitive test met the validity criteria of the test guideline detailed as follows (for the control group):

- the mortality of the parent animals (female Daphnia) did not exceed 20% at the end of the test (0%);

- the mean number of living neonates produced per parent animal surviving at the end of the test is > 60 (75.8)

In addition the CV (20.1%) of the mean number of living neonates produced per control parent animal surviving at the end of the test was lower than the threshold recommended by the OECD 211 guideline (25%).

Description of key information

Chronic toxicity test is available for invertebrates with di-tert-amyl peroxide: one OECD 211 GLP study is available with a 21 d NOEC of 1.2 mg/L and a 21d EC10 of 1.6 mg/L.

It was observed for the mean number of neonates produced per surviving parent.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.6 mg/L