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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-Sep-2010 to 17-Sep-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.
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 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.
Deviations:
no
Principles of method if other than guideline:
The test was performed as a limit test to demonstrate that the test item has no toxic effect on the test organisms up to a loading rate of 1000 mg/L.
GLP compliance:
yes (incl. QA statement)
Remarks:
swissmedic: Date of inspection: 05 to 09-Nov-2007 and 26 to 30-Nov-2007; Date of decision: 2008-04-30, Date of signature: 12-Nov-2008
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
For the determination of the actual test item concentrations of test part 1 (semi-static parent test), duplicate samples were taken from each treatment at the start and end of each test medium renewal period.
For the determination of the actual test item concentrations of test part 2 (static degradation products test), duplicate samples were taken from each treatment at the start and at the end of the test.
For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.
All samples were analyzed immediately after sampling. The concentrations of the test item Dilauroyl peroxide were analyzed in all duplicate test media samples from all sampling times. The duplicate control samples were also analyzed.

For further information on analytical procedures and results please see attached Appendix I - Analytical Investigations.
Vehicle:
no
Details on test solutions:
Due to the low water solubility of the test item (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNObel), dispersions of the test item were prepared. No auxiliary solvent or emulsifier was used.

Before weighing in the test item, the test item flakes were pestled.

Test part 1:
The dispersions were prepared just before the start of each test medium renewal period. The dispersions of the test item with the loading rate of 100 mg/L were prepared by dispersing 230.12 mg and 230.65 mg of the test item in 2300 mL of test water at test start and test medium renewal (after 24 hours), respectively. The dispersions of the test item with the loading rate of 1000 mg/L were prepared by dispersing 2.30075 g and 2.30096 g of the test item in 2300 mL of test water at test start and test medium renewal (after 24 hours), respectively.

The dispersions were intensely stirred for 72 hours at room temperature in the dark. During stirring the flasks were stoppered with glass stoppers and the air-space was kept at a minimum to avoid test item losses due to volatilization. The stirring period of 72 hours was chosen based on study MIRA20090934-water solubility, to dissolve a maximum amount of test item in the test water.

After the stirring period of 72 hours, undissolved test material was separated from the test water (as far as possible) using a separation funnel. The test item could not be filtered due to potential absorption of the test item to filter materials. Thus, the dispersions were left in the separation funnel to separate for about 30 minutes and the middle layers were used as test medium.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test part 2:
For test part 2 (degradation products test), the remaining volumes of the dispersions prepared at the start of test part 1 were used. They were left for ageing over a period of 7 days in a closed system to allow the formation of the eventual degradation products. Test part 2 was performed with the 7-day old test media.

The control medium (test water without test item) for test part 2 was also left for ageing over a period of 7 days in a closed system.
Test organisms (species):
Daphnia magna
Details on test organisms:
The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests (see below).

During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).

At the start of the test, the organisms used in the test were 6 to 24 hours old and were not first brood progeny.
Test type:
other: Test part one was conducted with a semi-static test design, while test part two was conducted under static conditions
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
not applicable
Hardness:
2.5 mmol/L
Test temperature:
The water temperature was maintained at 21 °C during the test ( for further information please see attached Table 7).
pH:
At the beginning and at the end of the test, the pH of the test media was 7.8 (for further information please see attached Table 7).
Dissolved oxygen:
The dissolved oxygen concentrations in the test media and control were at least 8.3 mg/L (for further information please see attached Table 7).
Salinity:
according to ISO 6341 mediu.
Nominal and measured concentrations:
Results of test part 1:
At the start of the test medium renewal periods of test part 1 (parent test), test item concentrations at the nominal test concentration of 100 mg/L were 1.4 and 0.25 mg/L. At the nominal test concentration of 1000 mg/L, concentrations of 9.2 and 13 mg/L were measured. At the end of the test medium renewal periods, test item concentrations at the loading rate of 100 mg/ were 0.44 and 0.26 mg/L. At the loading rate of 1000 mg/L, test item concentrations of 8.6 mg/L were determined.

Results of test part 2:
At the start of test part 2, the analytically determined concentrations of Dilauroyl peroxide in the test media of the loading rates of 100 and 1000 mg/L were 0.36 and 6.6 mg/L, respectively. At the end of the test, the analytically determined concentrations in the test media of the loading rates of 100 and 1000 mg/L were 0.95 and 5.6 mg/L, respectively.
Details on test conditions:
Test Water:
Reconstituted test water according to ISO 6341 was used in the study. For further information on test water please see section " Any other information on materials and methods incl. tables" .

Material:
As the test item was assumed to volatilize from water (Vapour pressure at 20 °C: 1.6 x 10-4 Pa according to Akzo Document n° “2.420.007 –“, resulting Henry constant based on a water solubility of 0.06 mg/L: 1.06 Pa.m3/mol), the test was performed in 50-mL glass tubes completely filled with about 60 mL of test medium and tightly closed with glass stoppers. The test tubes were labeled with the study number and all necessary additional information to ensure unique identification.

Experimental Conditions:
The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21°C (test parts 1 and 2).

A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was approximately between 520 and 680 Lux (test parts 1 and 2).

The daphnids were not fed during the test (test parts 1 and 2).

Study Design:
At request of the Sponsor, the test consisted of two parts: the test item Dilauroyl peroxide was tested in part 1; the test item and eventual degradation products of the test item were tested in part 2.

Two concentrations above the water solubility limit were tested. Therefore, test water was loaded with 100 and 1000 mg/L. Both loading rates resulted in measured concentrations above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNobel).

Control groups (test water without test item) were tested in parallel.

Test part 1 (parent test) was performed semi-static with test medium renewal after 24 hours to keep the concentrations of the test item Dilauroyl peroxide in the test media as constant as possible during the test period of 48 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly prepared test medium of the corresponding concentration.

Test part 2 (degradation products test) was performed static without test medium renewal.

For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnia was about 10 mL. Thus, the requirement of the test guidelines for the minimum volume of 2 mL test medium per daphnia was fulfilled. The daphnids were randomly distributed to the test vessels at initiation of the test.
Reference substance (positive control):
yes
Remarks:
Tested twice a year
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 9.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
>= 9.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 9.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 9.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Influence of the test item Dilauroyl peroxide on the mobility of Daphnia magna (test part 1):

At the start of the test medium renewal periods of test part 1 (parent test), test item concentrations at the nominal test concentration of 100 mg/L were 1.4 and 0.25 mg/L. At the nominal test concentration of 1000 mg/L, concentrations of 9.2 and 13 mg/L were measured. At the end of the test medium renewal periods, test item concentrations at the loading rate of 100 mg/ were 0.44 and 0.26 mg/L. At the loading rate of 1000 mg/L, test item concentrations of 8.6 mg/L were determined.

For each test medium renewal period, mean measured test concentrations were calculated as the geometric mean of the test item concentrations measured at the start and at the end of the test medium renewal period. Thereof, a mean measured test item concentration for the entire exposure period of 48 hours was calculated as the arithmetic mean of the two geometric means:

Loading rate
(mg/L) Mean measured test item concentration


100 0.55 mg/L
1000 9.7 mg/L

The mean measured test item concentrations were above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, Akzo Nobel). Thus, the mean measured test item concentrations result from dissolved and undissolved test material.

The biological results were related to the loading rates and to the mean measured test item concentrations (for further information please see attached Table 1).

In the control and at the loadings rates of 100 and 1000 mg/L, no immobilized test organisms were observed during the test period of 48 hours.

Therefore, the 48 hour NOEC and 48-hour EC0 of Dilauroyl peroxide to Daphnia magna were determined to be at least the loading rate of 1000 mg/L, corresponding to a mean measured test item concentration of 9.7 mg/L. The NOEC based on the mean measured concentration lies above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, Akzo Nobel).

The 48-hour EC50 and the 48-hour EC100 were clearly higher than the loading rate of 1000 mg/L. These values could not be quantified due to the absence of toxicity of Dilauroyl peroxide at the loading rate of 1000 mg/L.

Concerning the appearance of the test media, test item was floating on the surface of the test media of 100 and 1000 mg/L throughout the entire test period. Thus, undissolved test material could only be partially separated from the test media during media preparation.

At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.8 and 7.9 (for further information please see attached Table 3). The dissolved oxygen concentrations in the test media and control were at least 8.4 mg/L (for further information please see attached Table 4), and the water temperature was maintained at 21 °C during the test.

The test is considered to be valid, as in the control, no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.


Influence of eventual degradation products of the test item on the mobility of Daphnia magna (test part 2):

At the start of test part 2, the analytically determined concentrations of Dilauroyl peroxide in the test media of the loading rates of 100 and 1000 mg/L were 0.36 and 6.6 mg/L, respectively. At the end of the test, the analytically determined concentrations in the test media of the loading rates of 100 and 1000 mg/L were 0.95 and 5.6 mg/L, respectively.

The mean measured test item concentrations were calculated as the geometric means of the concentrations measured at the start and at the end of the test (test part 2):

Loading rate
(mg/L) Mean measured concentration
(mg/L)
100 0.58
1000 6.1


The biological results were related to the loading rates of the test item.

In the control and at the loadings rates of 100 and 1000 mg/L, no immobilized test organisms were observed during the test period of 48 hours (for further information please see attached Table 5).

Therefore, the 48 hour NOEC and 48-hour EC0 of Dilauroyl peroxide and its eventual degradation products were determined to be at least the loading rate of 1000 mg/L.

The 48-hour EC50 and the 48-hour EC100 were clearly higher than the loading rate of 1000 mg/L. These values could not be quantified due to the absence of toxicity of Dilauroyl peroxide and its degradation products at the loading rate of 1000 mg/L.

Concerning the appearance of the test media, test item was floating at the surface and lying on the bottom of the test vessels of the test media of 100 and 1000 mg/L throughout the test period (for further information please see attached Table 6).

At the beginning and at the end of the test, the pH of the test media was 7.8 (for further information please see attached Table 7). The dissolved oxygen concentrations in the test media and control were at least 8.3 mg/L, and the water temperature was maintained at 21 °C during the test.

Results with reference substance (positive control):
For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test of March 2010 (48-hour EC50: 0.43 mg/L, study C86933) proved the sensitivity of the test organisms and the validity of the test design (internal historical range of 48-hour EC50 from 2000 to 2010: 0.43 to 1.1 mg/L).
Reported statistics and error estimates:
Test part 1:
The 48 hour NOEC and 48-hour EC0 of Dilauroyl peroxide to Daphnia magna were determined to be at least the loading rate of 1000 mg/L, corresponding to a mean measured test item concentration of 9.7 mg/L. The NOEC based on the mean measured concentration lies above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, Akzo Nobel).

The 48-hour EC50 and the 48-hour EC100 were clearly higher than the loading rate of 1000 mg/L. These values could not be quantified due to the absence of toxicity of Dilauroyl peroxide at the loading rate of 1000 mg/L.
Therefore, the 48 hour NOEC and 48-hour EC0 of Dilauroyl peroxide and its eventual degradation products were determined to be at least the loading rate of 1000 mg/L.

Test part 2:
The 48-hour EC50 and the 48-hour EC100 were clearly higher than the loading rate of 1000 mg/L. These values could not be quantified due to the absence of toxicity of Dilauroyl peroxide and its degradation products at the loading rate of 1000 mg/L.

Test part 1:

For each test medium renewal period, mean measured test concentrations were calculated as the geometric mean of the test item concentrations measured at the start and at the end of the test medium renewal period. Thereof, a mean measured test item concentration for the entire exposure period of 48 hours was calculated as the arithmetic mean of the two geometric means:

 

Loading rate

(mg/L)

Mean measured test item concentration

 

100

0.55 mg/L

1000

9.7 mg/L

Test part 2:

The mean measured test item concentrations were calculated as the geometric means of the concentrations measured at the start and at the end of the test:

 

Loading rate
(mg/L)

Mean measured concentration
(
mg/L)

100

0.58

1000

6.1

Validity criteria fulfilled:
yes
Conclusions:
The test item Dilauroyl peroxide and its eventual degradation products had no acute toxic effects on Daphnia magna at test item loading rates and mean measured test item concentrations far above the water solubility limit of the test item under the conditions of the test.
Executive summary:

The influence of the test item Dilauroyl peroxide and its eventual degradation products on Daphnia magna was determined in a 48‑hour test according to the EU Commission Directive 92/69/EEC, Part C.2, the Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

 

The test item Dilauroyl peroxide was dispersed in test water at loading rates of 100 and 1000 mg/L. The dispersions were stirred for 72 hours in order to dissolve a maximum amount of the test item in the test water. After the stirring period, the dispersions were let to settle in a separation funnel and the middle layers were used as test media.The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

Half of the volumes of the test media were used immediately to examine the influence of the test item Dilauroyl peroxide on the mobility of Daphnia magna in a semi-static 48‑hour test (test part 1). The remaining volumes of the test media were left in a closed system for ageing over a period of 7 days in order to allow the test item to degrade and to test the influence of eventual degradation products of the test item on the mobility of Daphnia magna in a static 48‑hour test with 7-days aged test media (test part 2).

 

Control groups were run in parallel. 

The control medium for the degradation products test (test part 2) was left in a closed system for ageing over a period of 7 days.

 

Results of test part 1:

At the start of thetest medium renewal periods of test part 1 (parent test), test item concentrations at the nominal test concentration of 100 mg/L were 1.4 and 0.25 mg/L. At the nominal test concentration of 1000 mg/L, concentrations of 9.2 and 13 mg/L were measured. At the end of the test medium renewal periods, test item concentrations at the loading rate of 100 mg/ were 0.44 and 0.26 mg/L. At the loading rate of 1000 mg/L, test item concentrations of 8.6 mg/L were determined.

 

For each test medium renewal period, mean measured test concentrations were calculated as the geometric mean ofthe test item concentrations measured at the start and at the end of the test medium renewal period. Thereof, a mean measured test item concentration for the entire exposure period of 48 hours was calculated as the arithmetic mean of the two geometric means:

 

Loading rate

(mg/L)

Mean measured test item concentration

 

100

0.55 mg/L

1000

9.7 mg/L

The mean measured test item concentrations were above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNobel). Thus, the mean measured test item concentrations result from dissolved and undissolved test material.

 

The biological results of the Dilauroyl peroxide parent test (test part 1) were related to the loading rates and to the mean measured test item concentrations.

 

In the control and at the loadings rates of 100 and 1000 mg/L, no immobilized test organisms were observed during the test period of 48 hours. 

Therefore, the 48‑hour NOEC and 48-hour EC0of Dilauroyl peroxide to Daphnia magna were determined to be at least the loading rate of 1000 mg/L,corresponding to a mean measured test item concentration of 9.7 mg/L.

 

Results of test part 2:

At the start of test part 2, the analytically determined concentrations of Dilauroyl peroxide in the test media of the loading rates of 100 and 1000 mg/L were 0.36 and 6.6 mg/L, respectively. At the end of the test, the analytically determined concentrations in the test media of the loading rates of 100 and 1000 mg/L were 0.95 and 5.6 mg/L, respectively.

 

The mean measured test item concentrations were calculated as the geometric means of the concentrations measured at the start and at the end of the test (test part 2):

 

Loading rate
(mg/L)

Mean measured concentration
(
mg/L)

100

0.58

1000

6.1

 

The biological results of the degradation products test (7-days aged test media, test part 2) were related to the loading rates of the test item.

 

In the control and at the loadings rates of 100 and 1000 mg/L, no immobilized test organisms were observed during the test period of 48 hours.

 

Therefore, the 48‑hour NOEC and 48-hour EC0of Dilauroyl peroxide and its eventual degradation products were determined to be at least the loading rate of 1000 mg/L.

 

Description of key information

Using the WAF approach with analytical determination of the test substance, neither the substance nor potential degradation products had any effects up to or above the limit of solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1 000 mg/L

Additional information

2 studies on daphnids are available, one invalid study (Gancet, 2008a) which was performed using filtered stock solutions, perhaps removing any dissolved substance present, and a reliable GLP study (Weber, 2010a)

. This study tested the influence of the test substance which is poorly soluble and its degradation products on daphnid immobilisation under closed conditions. Briefly two stock solutions were first prepared at loading rates of 1 mg/L and 100 mg/L with test media. Stock solutions were stirred for 72h (the time believed from preliminary studies to be the optimal time for maximising the parent compound concentration in aqueous solution) and then left in a separation funnel for Phase separation. The stock solutions were then split into two batches: one batch for direct use and a second batch aged for 7 days, in order to allow hydrolysis products of the substance to be present should it be likely to degrade abiotically. After 48h of exposure, mean measured concentrations in the two tests were respectively 9.7 mg/L for the stock solution at a loading rate of 1000 mg/L for the parent substance test and 6.1 mg/L for the stock solution at a loading rate of 1000 mg/L in the degradation product test. Both of these concentrations are known to be well over the aqueous solubility limit of the substance. No effects were observed in either of the groups (parent or potential degradation products tested). It was concluded that neither the substance nor potential degradation products had any effects up to the limit of solubility.