Dihexadecyl peroxodicarbonate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

December 2020 - October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please refer to read-across document attached.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was pre-dissolved in acetone. Aliquots of the acetonic solution were applied to the test medium. Due to instability of the analyte in solutions, stock solutions were prepared freshly for each day of application. For each concentration step, an own acetonic stock solution was prepared. An aliquot of each stock solution was transferred to 20 L of dilution water and stirred for 2h. From this application solution, 5 L were transferred to each test vessel.
- Controls: yes, blank and solvent
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: 200 µL
- Test concentration separation factor: 1.59
- Evidence of undissolved material: no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Danio rerio, Teleostei, Cyprinidae)
- Source: Test facility bred
- Origin of the used strain: West Aquarium GmbH, 37431 Bad Lauterberg, Germany

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Eggs were collected in a glass spawning-tray, which were placed at the bottom of the holding vessels. The tray was covered with a stainless-steel lattice to prevent adult fish from predating the eggs. An artificial substrate (modified method according to Nagel, R (1986)) was attached to the lattice to stimulate spawning into the tray. Turning on the lighting (one neon lamp per vessel, light intensity approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel) induced mating and spawning of fish.

- Subsequent handling of eggs: The collected eggs were transferred from the spawning-tray into a sieve, rinsed with clean water in order to remove any debris and then put into glass dishes. Fertilized eggs (microscopic determination of >four cell stage, i.e. early blastula stage) were then transferred by means of a widened and de-burred pipette tip into the test chambers. Time from spawning until transferring into the test solutions was kept as short as possible.

POST-HATCH FEEDING
- Start date: 5 dpf
- Type/source of feed: ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany) and liquid rearing feed (Nobil fluid, JBL, Neuhofen, Germany).
- Frequency of feeding: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Hardness:

1.0 - 1.1 mmol/L

Test temperature:

25.5 - 27.1 °C

pH:

7.52 - 8.10

Dissolved oxygen:

73 - 106 %

Conductivity:

251-257 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 50, 80, 126, 201 and 320 µg/L
Measured concentrations: 111, 184, 235, 293 and 403 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: tanks
- Type: open
- Material, size, headspace, fill volume: glass, 6.5 L, -, 5 L test medium
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): every 48/72 h
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: <0.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water
- Total organic carbon: 1.070 - 1.104 mg/L
- Chlorine: 0.02 mg/L
- Alkalinity: 1.6 - 1.8 mmol/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: Light/dark cycle - 12 h/12 h
- Light intensity: approximately 1000 lux

EFFECT PARAMETERS MEASURED: hatching success, post-hatch survival, length, weight

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: up to 200 µg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatch success

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatch success

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

>= 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

>= 403 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Test conditions – Water quality parameters

The water temperature was in the range of 25.5 °C to 27.1 °C in all test vessels throughout the in-life phase of the study and did not differ by more than 1.5 °C on successive days, and thus is in line with the guideline requirements. The oxygen saturation in all test vessels was between 73 % and 106 % throughout the study. The pH in the test vessels was between 7.52 and 8.10.

 

Test item concentrations

The concentrations of MYPC were assessed by chemical analysis using LC-MS. The LOQ was set to 10 µg MYPC/L. At test start, samples of fresh test solutions were taken from each test vessel before adding of test organisms. For aged test solutions, samples were taken also for all test vessels at the day of the medium renewal. Thereafter, sampling was performed once per week in fresh and aged test media.

For one renewal event per week, samples of fresh and aged test medium were taken from one test replicate of dilution water and solvent control, and furthermore from each treatment concentration applied.

Initial MYPC values (i.e. fresh medium samples) were considered for the calculation of the replicate arithmetic means. Based on this, the mean measured initial MYPC values per treatment were determined at 111, 184, 235, 293 and 403 µg/L. Thus, these concentrations were between 126 % (nominal concentration of 320 µg MYPC/L) and 230 % (nominal concentration of 80 µg MYPC/L).

The biological effect concentrations were based on arithmetic mean measured initial concentrations.

Rapid degradation of test item concentrations was observed, and was proven by the measurement of aged samples:

The mean measured MYPC concentrations of aged solution (24h) per treatment were determined at 15.4, 18.4, 43.4, 19.5 and 20.7 µg MYPC/L. These concentrations were between 6.48 % (nominal concentration of 320 µg MYPC/L) and 34.4 % (nominal concentration of 50.0 µg MYPC/L).

For two study dates, samples of test medium aged for 48 h were taken and measured, i.e day 2 and day 23 of the study. All of the 48h samples, except of one sample, were below the limit of quantification. The single sample above the limit of quantification was at 14.9 % of the nominal concentration of 320 µg MYPC/L.

 

Biological endpoints

There was no statistically significant difference between water control and solvent control for any endpoint assessed. In accordance with the OECD test guideline 210, the two controls were compared for each response and combined for statistical analysis.

 

Hatching rate

First larvae hatched on day 2 post fertilization (pf) in the controls and the treatment level. Hatch was completed at day 5 pf in all repliactes, with no difference between the treatments and the controls. The mean values on hatch are shown in Table 3.

 

Table 3:        Biological effects during larval development; Hatching rate between day 2 pf and day 5 pf

		Nominal concentration MYPC [µg/L]
		Dilution Water control	Solvent control	50	80	126	201	320
		Mean measured initial concentration MYPC [µg/L]
		Dilution Water control	Solvent control	111	184	235	293	403
No of replicates		4	4	4	4	4	4	4
Hatch, day 2 pf [%]	Mean	22.5	7.5	1.3	1.3	6.3	1.3	5.0
	SD	18.5	9.6	2.5	2.5	6.3	2.5	7.1
	RSD [%]	82.2	127.7	200.0	200.0	100.7	200.0	141.4
Hatch, day 3 pf [%]	Mean	63.8	41.3	38.8	30.0	35.0	40.0	58.8
	SD	16.5	19.3	15.5	15.8	17.8	4.1	4.8
	RSD [%]	25.9	46.8	39.9	52.7	50.8	10.2	8.1
Hatch, day 4 pf [%]	Mean	100.0	100.0	100.0	98.8	100.0	100.0	100.0
	SD	0.0	0.0	0.0	2.5	0.0	0.0	0.0
	RSD [%]	0.0	0.0	0.0	2.5	0.0	0.0	0.0
Hatch, day 5 pf [%]	Mean	100.0	100.0	100.0	100.0	100.0	100.0	100.0
	SD	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	RSD [%]	0.0	0.0	0.0	0.0	0.0	0.0	0.0

SD = Standard deviation

RSD = Relative standard deviation

 

Post hatch survival

The number of surviving fish larvae was determined at day 21 pf and at test end at day 35 pf by photo recording and evaluation. At test end, the mean post hatch survival rate was 82.5 % in the water control and 83.8 % in the solvent control. Thus, water- and solvent-control both met the validity criterion for survival in controls of ≥75 %. The post hatch survival was 78.8, 78.8, 87.5, 83.8 and 83.8 in the mean measured initial concentrations of 111, 184, 235, 293 and 403 µg MYPC/L, respectively. Statistical analysis revealed no significant difference in survival between control and treatments (Dunnett t-test, α = 0.05, one-sided smaller).

 

Table 4:        Biological effects during larval development; Post hatch survival at day 21 pf and day 35 pf

		Nominal concentration MYPC [µg/L]
		Dilution water control	Solvent control	50	80	126	201	320
		Mean measured initial concentration MYPC [µg/L]
		Dilution water control	Solvent control	111	184	235	293	403
No of replicates		4	4	4	4	4	4	4
Post hatch survival, at day 21 pf [%]	Mean	83.8	83.8	78.8	78.8	87.5	83.8	86.3
	SD	9.5	13.8	10.3	17.0	11.9	18.0	21.0
	RSD [%]	11.3	16.4	13.1	21.6	13.6	21.5	24.3
Post hatch survival, at day 35 pf [%]	Mean	82.5	83.8	78.8	78.8	87.5	83.8	83.8
	SD	8.7	13.8	10.3	17.0	11.9	18.0	19.3
	RSD [%]	10.5	16.4	13.1	21.6	13.6	21.5	23.1

SD = Standard deviation

RSD = Relative standard deviation

 

Growth

At test end, larval growth in terms of total length, wet weight and dry weight were determined. The test guideline [8] mentions a typical minimum total length for control larvae of 11 mm. The mean individual total length of the fish at test end was 15.2 mm in both, dilution water- and solvent control, while 14.9, 15.3, 14.9, 14.5 and 14.9 mm were measured in treatments of 111, 184, 235, 293 and 403 µg MYPC/L, respectively (Table 5). There was no statistically significant difference in individual total length of fishes exposed to the test item concentrations compared to the pooled controls (Dunnett´s multiple t-test, α = 0.05, two-sided).

Fish growth was also determined in terms of individual dry weight. The mean individual dry weight of the fish at test end was 6.8 mg in the water control and 6.9 mg in the solvent control, while 7.1, 7.2, 6.5, 6.5 and 6.6 mg were measured in treatments of 111, 184, 235, 293 and 403 µg MYPC/L, respectively (Table 5). There was no statistically significant difference in individual dry weight of fishes exposed to the test item concentrations compared to the pooled controls (Dunnett t-test, α = 0.05, two-sided).

 

Table 5:        Biological effects during larval development; Growth at 35 dpf (test end)

		Nominal concentration MYPC [µg/L]
		Dilution Water control	Solvent control	50	80	126	201	320
		Mean measured initial concentration MYPC [µg/L]
		Dilution Water control	Solvent control	111	184	235	293	403
No of replicates		4	4	4	4	4	4	4
Total length, at day 35 pf (test end) [mm]	Mean	15.2	15.2	14.9	15.3	14.9	14.5	14.9
	SD	0.5	0.2	0.7	0.4	0.2	0.2	0.6
	RSD [%]	3.1	1.6	4.4	2.9	1.5	1.7	3.7
Individual fish dry weight, at day 35 pf (test end) [mg]	Mean	6.8	6.9	7.1	7.2	6.5	6.5	6.6
	SD	0.7	0.4	0.9	1.1	0.1	0.5	0.9
	RSD [%]	10.1	5.4	12.7	15.4	2.2	7.3	14.1

SD = Standard deviation

RSD = Relative standard deviation

Validity criteria fulfilled:

yes

Conclusions:

Statistical evaluation revealed no significant effect of MYPC on hatchability, post hatch survival or growth in terms of individual total length and individual dry weight up to the highest tested concentration of 403 µg/L (mean measured concentration).

Executive summary:

An early life stage toxicity test with zebrafish (Danio rerio), sponsored by United Initiators GmbH, was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME). The aim of the study was to derive a NOEC (no observed effect concentration) and to calculate EC_{10, 20, 50} (Effective concentration which results in a 10, 20 or 50 % reduction in the measured parameter), if possible. For this reason, early life stages of zebrafish (Danio rerio) were exposed to five concentrations of the test item, a solvent control and a dilution water control under semi-static conditions for a period of 35 d (according to Annex 2 of OECD guideline 210). Renewals of test media were performed three times per week.

The study was performed at five nominal test concentrations (50, 80, 126, 201 and 320 µg MYPC/L). All treatments were applied in four replicates each. The test started with 20 randomly introduced eggs. Hatching rate, mortality, growth, and behavioral abnormalities were recorded.

 

Chemical analysis

The concentrations of MYPC were assessed by chemical analysis using LC-MS. The LOQ was set to 10 µg MYPC/L. At test start, samples of fresh test solutions were taken from each test vessel before adding of test organisms. For aged test solutions, samples were taken also for all test vessels at the day of the medium renewal. Thereafter, sampling was performed once per week in fresh and aged test media.

For one renewal event per week, samples of fresh and aged test medium were taken from one test replicate of dilution water and solvent control, and furthermore from each treatment concentration applied.

Initial measured MYPC values (i.e. fresh medium samples) were considered for the calculation of the replicate arithmetic means. Based on this, the mean initial measured MYPC values per treatment were determined at 111, 184, 235, 293 and 403 µg/L. Thus, these concentrations were between 126 % (nominal concentration of 320 µg MYPC/L) and 230 % (nominal concentration of 80 µg MYPC/L).

The biological effect concentrations were based on arithmetic mean measured initial concentrations.

Rapid degradation of test item concentrations was observed.

 

Biological effects

 

Hatching success and post hatch survival

First larvae hatched day 2 post fertilization (pf). Hatch was completed at day 5 pf, in all replicates, with no difference between treatments. No coagulated eggs were found, thus hatching success was 100 % across all treatments.

The occurrence of fish mortality for larvae and juveniles was observed daily. The number of surviving fish was recorded at day 21 pf and at test end at day 35 pf by photo recording and evaluation. Mortality of larvae occurred mainly before day 21 pf, during the phase of transition from yolk sac feeding to external feeding.

At test end, the mean post hatch survival rate was 82.5 % in the water control and 83.8 % in the solvent control. Thus, both water- and solvent-control met the validity criterion for survival in controls of ≥75 % as required by the guideline.

At test end, the post hatch survival in treatments was determined to be 78.8, 78.8, 87.5, 83.8 and 83.8 % in the test concentrations of 111, 184, 235, 293 and 403 µg MYPC/L (mean measured initial concentrations), respectively. Statistical analysis did not show any significant differences in survival between controls and treatments (Dunnett´s multiple t-test, α = 0.05, one-sided smaller).

Due to the absence of a concentration related effect, the calculation of an ECx value for the endpoint post hatch survival was not possible and thus under the test conditions, the EC_{10, 20, 50} are estimated to be above the highest concentration.

 

Individual total length at test termination

The mean individual total length of the fish at test end was 15.2 mm in both, dilution water and solvent control, while 14.9, 15.3, 14.9, 14.5 and 14.9 mm were measured in treatments of 111, 184, 235, 293 and 403 µg MYPC/L.

Statistical analysis did not show any significant differences in individual total length between control and treatments (Dunnett´s multiple t-test, α = 0.05, two-sided).

Due to the absence of a concentration related effect, the calculation of an ECx value for the endpoint total length was not possible and thus under the test conditions, the EC₁₀, ₂₀, ₅₀ are estimated to be above the highest concentration.

 

Individual dry weight at test termination

At test end, the mean individual dry weight was 6.8 mg in the water control and 6.9 mg in the solvent control. In treatments the mean individual dry weight was assessed to be 7.1, 7.2, 6.5, 6.5 and 6.6 mg in test item concentrations of 111, 184, 235, 293 and 403 µg MYPC/L (mean measured initial concentrations), respectively.

Statistical analysis did not show any significant differences in individual dry weight between control and treatments (Dunnett´s multiple t-test, α = 0.05, two-sided).

Due to the absence of a concentration related effect, the calculation of an ECx value for the endpoint dry weight was not possible and thus under the test conditions, the EC₁₀, ₂₀, ₅₀ are estimated to be above the highest concentration.

 

Behaviour

No abnormal behavior of the fish was observed during the study.

 

Conclusion

Statistical evaluation revealed no significant effect of MYPC on hatchability, post hatch survival or growth in terms of individual total length and dry weight. For all parameters, under the test conditions applied, the EC₁₀, ₂₀, ₅₀ are estimated to be above the highest concentration.

All effect concentrations obtained from the study are shown in the following Table.

 

Table 1:          Summary of statistical evaluation during the course of the study

Parameter	NOEC / LOEC Nominal concentration MYPC	NOEC / LOEC Mean measured initial concentration MYPC
	[µg/L]	[µg/L]
Hatching success	≥320 / >320	≥403 / >403
Post hatch survival at day 21 pf *)	≥320 / >320	≥403 / >403
Post hatch survival at day 35 pf *)	≥320 / >320	≥403 / >403
Individual total length at day 35 pf **)	≥320 / >320	≥403 / >403
Individual dry weight at day 35 pf **)	≥320 / >320	≥403 / >403

*)   Dunnett´s multiple t-test, α = 0.05, one-sided smaller

**) Dunnett´s multiple t-test, α = 0.05, two-sided

Description of key information

The toxicity of the closely related substance Dimyristiylperoxydicarbonate with CAS 5322-14-5 was tested in the Early-Life Stage Toxicity Test with Danio rerio under semi-static conditions according to OECD 210 (2013). Fish were exposed for 35 days to a range of test concentrations up to 320 µg/L, nominal (403 µg/L mean measured). 

Statistical evaluation revealed no significant effect of the test substance on hatchability, post hatch survival or growth in terms of individual total length and individual dry weight up to the highest tested concentration of 403 µg/L (mean measured concentration).

Therefore NOEC >= 403 µg/L, which is far above the maximum water solubility of < 1 µg/L.

 

For additional information see the accompanying read-across document. Due to the physical chemical properties of these substances being similar and the similarity in aquatic toxicity profile it is considered justified to read across to the existing long-term fish endpoint for Dimyristiylperoxydicarbonate. This study has therefore been highlighted as the key information for this endpoint. 

Additional information

The study was conducted with the read-across substance 53220-22-7.

A correction has to be made still on the calculated mean measured concentration, which will be done in a report amendment. The arithmatic mean was used instead of the geometric mean. The geometric mean will be slightly lower than the arithmatic mean reported now. Still endpoints will be far above the maximum water solubility of this registered substance, Dicetyl peroxydicarbonate. The maximum water solubility could not be measured accuratly in the OECD 105 study, since it was below LOD of 1 µg/L. Therefore, the conclusion of the OECD 210 study will still be that there are no effects up to the water solubility limit and the study result can be used as is.