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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:
2000-10-31 to 2001-06-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
According to method C2 of the European Directive 92/69/EEC which is in conformity with the OECD guideline 202 (part I)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the beginning of the 1st definitive test and prior to addition of the organisms, test solutions are sampled and chemically analysed to verify that initial concentrations are equivalent to nominal concentrations. Chemical analysis of test solution samples is also performed at the end of the test.
- The tested item is a mixture of 2 constituents with different physico­chemical properties. Test solutions were prepared according to the WAF methodology (Water Accommodated Fraction) described by OECD.
- Concentrations: first definitive test (loading rate (WAF)): 0, 30.3, 40.8, 55, 74.9, 99.7 mg/L
- Sample storage conditions before analysis: cold and dark
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances) - 2nd definitve test
- Method:
Since cumylperoxyneodecanoate in isododecane is a preparation containing 2 chemicals having different physico-chemical properties, the preparation of test solutions was carried out using the WAF (Water Accomodated Fraction) methodology described by OECD. Individual solutions were prepared by vigorously mixing 3.9; 7.5; 16; 33 and 70.4 mg of cumylperoxyneodecanoate in isododecane with 1 litre of dilution water. After 66 hours of stirring, excess of non-dissolved test item was removed by filtration on Millipore membranes (HV 0.45 µm).
- Differential loading: (nominal concentrations): 3.9; 7.5; 16; 33 and 70.4 mg/L
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): excess of non-dissolved test item was removed by filtration on Millipore membranes (HV 0.45 µm).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain/clone: clone 5 or clone A
- Source: Animals come from a breeding stock in the testing laboratory
- Age of parental stock (mean and range, SD): < 24 hours old. Selection of test organisms is made by filtration.
- Feeding during test: not reported

ACCLIMATION: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
mobile Daphnia were counted after 24 and 48 h
Test temperature:
18.5 - 19.5 °C
pH:
7.3 - 7.9
Dissolved oxygen:
6.9 - 8.5 mg/L
Nominal and measured concentrations:
1. definitive test: Loading rate (WAF) nominal (mg/L): 0.0; 30.3; 40.8; 55.0; 74.9; 99.7
2. definitive test: Loading rate (WAF) nominal (mg/L): 0.0; 3.9; 7.5; 16.0; 33; 70.4 (relevant for calculation of ECx values)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass bottles
- Type (delete if not applicable): closed with butyl rubber caps covered with PTFE bungs and selaed with aluminium caps
- Material, size, headspace, fill volume: glass, 120 mL
- Renewal rate of test solution (frequency/flow rate): No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra-pure water was used for the preparation of dilution water (resistivity > 18MQ). Dilution water is prepared according to the protocol described in standard NF EN ISO 6341.
- The following stock solutions are prepared:
- 11.76 g of CaCl2, 2H2O per litre of water,
- 4.93 g of MgSO4, 7H2O per litre of water,
- 2.59 g of NaHCO3 per litre of water,
- 0.23 g of KCl per litre of water.
Dilution water is obtained by adding 25 ml of each of the stock solutions in a final volume of 1 litre and then aerating the solution until oxygen saturation.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity: testing flasks were incubated in darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation

RANGE-FINDING STUDY Yes
- Test concentrations: 0.1 - 100 mg/L
- Results used to determine the conditions for the definitive study: yes
- geometric progression factor: not exceeding 2.1
- For the preliminary test, a stock solution of the test item diluted in water was prepared 24 hours before the beginning of the test by mixing 100 mg of cumylperoxyneodecanoate in isododecane in 1 L of dilution water. Volumetric flasks were filled with the required volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 0.1 mg/L to 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%-C.I.: 100 - 440
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
4.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%-C.I.: 0.002 - 12
Details on results:
- Behavioural abnormalities: Not observed
- Mortality of control: 5 %
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation was observed at the end of the test.

Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1 mg/L

Control of the biological reagent sensitivity
The 92/69/EEC C.2. and OECD 202 methods do not require to test a reference item simultaneously to the assay on the test item. However, the sensitivity of the test system and the methodology were evaluated every two months by performing an Daphnia acute toxicity test on sodium dichromate. The most recent value of EC50 obtained on January 24, 2001 was 1 mg/L. For information, ISO 6341 has a validity criterion specifying that the EC50 must be in the range 0.6 to 1.7 mg/l.
Reported statistics and error estimates:
Results were obtained by regression analysis using the Probit/log model.

Analysis of solution during the first definitive test showed that:

- Cumylperoxyneodecanoate is not found in test solutions at time 0 and 48 h (concentration lower than LoQ which is equal at 0.25 mg/L for this method).

- Neodecanoic acid, a potential by-product of cumyl peroxyneodecanoate hydrolysis was found in the test solution at time 0 and time 48 h. The relative concentration of neodecanoic acid was found to be constant during the test period (see table 4).

- Concentration of neodecanoic acid: (nominal): 1st definitive test - 30.3, 40.8, 55, 74.9, 99.7 mg/L and 2nd definitive test 3.9; 7.5; 16.0; 33; 70.4 mg/L

- The final concentration of the sub-product neodecanoic acid were maintained within the designated limit of 80 % of the initial concentrations in the test solutions.

The percentage inhibition of Daphnia mobility are presented in the following tables:

Table 1. First definitive test

 Loading rate (WAF) (mg/L)     Immobilisation (%)
   24 h  48 h
 0  0  5
 30.3  5  85
 40.8  15  90
 55  35  100
 74.9  15  100
 99.7  100  100

Table 2.Second definitive test

  Loading rate (WAF) (mg/L)   Immobilisation (%)   
    24 h   48 h
 0  0  5
 3.9  0  45
 7.5  0  65
 16  0  55
 33  5  80
 70.4  20  85

Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test:

Table 3. Second definitive test

Loading rate (WAF) (mg/L)

pH

Dissolved O2 (mg/L)

T0

T48h

T0

T48h

0

7.7

7.8

8.6

8.6

3.9

ND

7.83

ND

7.8

7.5

ND

7.83

ND

7.8

16

ND

7.72

ND

8.8

33

ND

7.67

ND

8.2

70.4

7.65

7.59

8.1

8.3

The relative concentration of neodecanoic acid was found to be constant during the test period (see table 4). However, since no commercial standard of neodecanoic acid was available, the exact quantification of this sub-product was not carried out. The results are expressed as equivalents of an internal standard (lauric acid). They are used in order to correlate percentage of final over initial concentrations.

 

Table 4: Concentration of neodecanoic acid (expressed as equivalent in lauric acid) for the first definitve test

Nominal (mg/L)

Final/Initial (%)

99.7

99.3

74.9

99.6

55

99.7

40.8

99.0

30.3

97.4

 

As shown in the previous table, the final concentrations of the sub-product neodecanoic were maintained within the designated limit of 80 % of the initial concentrations in test solutions. Thus, the test item concentrations were maintained throughout the duration of the test.

The analytical results from the first definitive test proove that the nominal concentrations were within 80 % of the expected range and are stable over the test period. Hence, for the second test the nominal loadings were used.

Validity criteria fulfilled:
yes
Conclusions:
The determination of the inhibition of mobility (acute toxicity) of Daphnia magna exposed to the test item cumylperoxyneodecanoate in isododecane for a duration of 48 hours was performed following the method C2 described in Directive 92/69/EEC of the European Commission and in the guideline 202 (part I) of the OECD. The following effect level were calculated:
- 24-hr-EL50: 160 mg/L
- 48-hr-EL50: 4.9 mg/L
Executive summary:

The 48–hr acute toxicity of cumylperoxyneodecanoate (71.5 % in isododecane) to Daphnia magna Straus was studied under static conditions according to method C.2 of the European Directive 92/69/EEC which is in conformity with the OECD guideline 202 (part I). Two definitive tests were realised. The second definitive test was carried out since the results of the first one did not allow to get a good correlation of the effect/concentration relationship.

Daphnids were exposed to a control, and cumylperoxyneodecanoate in isododecane at nominal loadings of 3.9; 7.5; 16.0; 33.0 and 70.4 mg/L for 48 hr. The test item is a mixture of 2 constituents with different solubilities and physico­chemical properties. Therefore, test solutions were prepared according to the WAF methodology (water accommodated fraction) described by OECD. Immobilization was observed daily. 

Concentrations of the test item were measured for the first definitve test by HPLC coupled with mass spectrometry. Cumylperoxyneodecanoate was not found in test solutions at time 0 and 48 h. Therefore, the degradation product neodecanoic acid was analysed. The relative concentration of neodecanoic acid was found to be constant during the test period. Since no commercial standard of neodecanoic acid was available, the exact quantification of this sub-product was not carried out. The results are expressed as equivalents of an internal standard (lauric acid). They are used to correlate the percentage of final

over initial concentrations. The concentrations were maintained within the designated Iimit of 80 % of the initial concentrations in the test solution. Thus, effective concentrations have been calculated using the nominal loading of cumylperoxyneodecanoate in isododecane. Hence, for the second test the nominal loadings were used for calculation of ECx values.

The 48–hr EL50 was 4.9 mg/L.

Based on the results of this study, the test item would not be classified as toxic to D. magna in accordance with the CLP classification system.  

 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.  

 

Results Synopsis

 

Test Organism Age (e.g. 1st instar): 6 - 24 h

Test Type: Static

 

24–hr EL50:   160 mg/L                      95% C.I.:  100 - 440 mg/L

48–hr EL50:    4.9 mg/L                      95% C.I.:  0.002 - 12 mg/L(C.l.: confident interval)

 

Endpoint(s) Effected:  Immobility/Mortality

Description of key information

The 48-hr EL50 of 1-methyl-1-phenylethyl peroxyneodecanoate reported by Thiébaud & Kaé (2001) for Daphnia magna Straus under the test conditions of OECD 202 was 4.9 mg/L (nominal; peroxide content 71.5 %).

The test item is not stable in aqueous solutions at the test temperature and was not detectable at or below the limit of quantification (70 µg/L). Therefore, actual concentrations of the degradation product neodecanoic acid were analysed at the beginning and at the end of the test. As the concentration of neodecanoic acid was stable during the total test duration, stable composition of the test solutions is concluded.

A re-calculation of the effect value based on actual concentrations of neodecanoic acid results in a theoretic EC50 of 2.15 mg/L. The re-calculation was conducted by means of a conversion factor of 2.275. This factor was deduced from the measured neodecanoic acid concentrations in relation to nominal peroxide loadings presented in the study report. 

However, it is known that actual acute effect levels of neodecanoic acid to D. magna are well above 2.15 mg/L. It is concluded that the ecotoxicological effects noted in the study of Thiébaud & Kaé (2001) are related to unknown decomposition products of 1-methyl-1-phenylethyl peroxyneodecanoate. Therefore, the 50 % effect concentration of 2.15 mg/L was back-calculated to the parent substance based on the molecular weight ratio of 1-methyl-1-phenylethyl peroxyneodecanoate to neodeconoic acid (1.78):

48-hr EC50 = 3.8 mg/L.

In conclusion, for 1-methyl-1-phenylethyl peroxyneodecanoate the 48-hr EC50 of 3.8 mg/L to Daphnia magna Straus is carried forward in the hazard and risk assessment.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
3.8 mg/L

Additional information