1,1,3,3-tetramethylbutyl peroxyneodecanoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.5.2012-5.10.2012 (analytics repeated)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study to appropriate guideline. Analysis on degradation components not conducted. Analysis of parent component was conducted as a parallel analytical investigation in test medium not in the actual toxicity test due to an initial failure of the original analytical method. Methods slightly adapted but justified. Otherwise reliable guideline study with mentioned restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

WAF method used due to extremely low solubility

Principles of method if other than guideline:

For stability reasons the test chemical is mixed with Isododecane. Testing without the presence of Isododecane is not feasible for stability and safety reasons. The marketed product was therefore considered a multi component substance containing a mixture of individual substances with different solubility and physical chemical properties. The test chemical was therefore be tested as such, using the water accommodated fraction method (WAF). Furthermore the test material is instable and hydrolizes and thermically decomposes relitively rapidly to multiple degradation products. The WAF method therefore allows the hazard of the resulting test material as a whole to be determined.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling was intended for the start and for the end of the test. However the tests running in parallel had indicated that the test substance was
no longer detectable after 24 hours. For this reason only two samples were analyzed. 0 (representative of the start of the test after stirring period) and 24 hours representative of the first 24 hours of the test.

Vehicle:

no

Details on test solutions:

Culture medium was prepared by diluting the OECD stock mineral salts in an appropriate vessel with de-ionized water. The medium was then sterilized by filter sterilization. The WAF solutions at the desired test concentrations were then prepared separately, sealed and left to stir slowly for approximately 3 days and then stand for approximately 1 hour. After which each solution was individually siphoned into a centrifuge tube and centrifuged at 8000 RPM for 10 minutes at 20 ºC. The resulting solutions were transferred to the test vessels from the middle of the centrifuge tube avoiding as far aspossible the transfer of surface film and/or un-dissolved test substance. The test vessels were then inoculated with the test organism and sealed with a cotton wool stopper and incubated for 72 hours. Absorbance was measured spectrophotometrically at 436 nm after 0, 24, 48 and 72 hours to allow determination of the desired endpoints.


Preparation of solutions
Due to the test substance being instable and poorly soluble an extended time is required to reach a stable concentration of the parent component in water. This was demonstrated in water solubility studies. An appropriate preparation method is therefore required whilst bearing in mind extended stirring means greater accumulation of degradation products via hydrolysis and thermal degradation.

A WAF method with a 3 day stirring time mimicking the conditions used in the solubility test was used for this study. This allowed both the maximum achievable concentration of parent and any degradation products generated in this period the possibility to dissolve up to their solubility limit in the test media. Thus assessing the direct effect the test material as a whole has on the test organism.

A traditional stock solution and subsequent dilutions were therefore not made during this test.

A WAF was prepared for each test concentration individually by adding of an accurate amount of the test substance to the test media and allowing equilibrate under slow agitation over approximately 3 days. Each WAF was then considered loaded with the test substance components and / or breakdown products at their corresponding water solubility limits. The WAF solutions were then centrifuged as previously described and the resulting solutions were transferred to the test vessels.

The inoculum was then added to each test vessel from an exponentially growing culture and the test vessel was sealed and incubated for the test duration. In addition, 6 control replicates were tested containing test media only. The extinction of the contents of each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Small amounts of un-dissolved test substance were still visible in the test vessels of the higher concentrations despite best efforts to prevent this. A higher than expected concentration may have been present in these vessels.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK. After purchasing, this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase. For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Observed after test for bacterial contamination only

Hardness:

Standard reccomended OECD algae media used

Test temperature:

22.2 to 22.9 °C during the test period

pH:

Media = pH 8.0 -8.2
Variation during test in control pH= 8.0-9.3

Dissolved oxygen:

not applicable

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations were 8.0,25.6, 81.9, 262.1 and 838.4 mg/L
Measured concentration of parent concentration in parallel analytical study shown in table below .

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer 100ml
- Type (delete if not applicable): open
- Material, fill volume: 40 Ml
- Aeration:No
- Initial cells density:As per guideline
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium used without modification

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod:72h
- Light intensity and quality:As per guideline


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber (periodically); spectrophotometer during test]
-

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study- multiple studies conducted see supporting study

Reference substance (positive control):

yes

Remarks:

Pottasion Dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

104.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

200.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Due to the parent chemical being measured at similar concentrations regardless of loading it can be concluded that toxicity observed here was due to hydrolytic and thermal degradation products. The toxicity observed is very low and would not result in classification of the product. Chemical analysis demonstrated the presence of and subsequent rapid disappearance of the parent chemical. Due to the complex mixture formed endpoints have been expressed as loadings of the test material.

Results with reference substance (positive control):

Conducted periodically on the test strain as part of the GLP maintainance. Test culture acceptable for use.

Reported statistics and error estimates:

The mean values of the observed absorbance for each test substance concentration were used to calculate the growth and specific growth rate. Details of the calculations are given in Annex 2. Where possible, the EC10,50 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50, whereas the EC50 value cal¬culated for the specific growth rate is termed ErC50. The LOEC was determined by comparison of the growth at each concentration and the control using the Dunnett’s test. The NOEC was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. All computations were performed using the TOXCALC version V 5.0.23 program.

Measured test substance concentrations (Stability test without test organisms)

Name	Nominal Conc. (mg/L)	Measured conc. (mg/L)	Final conc. (µg/L)
T11032 Recovery OECD I-1 T=0	8.0	0.0148	14.8
T11032 Recovery OECD I-2 T=0	8.0	0.0151	15.1
T11032 Recovery OECD II-1 T=0	81.9	0.0167	16.7
T11032 Recovery OECD II-2 T=0	81.9	0.0157	15.7
T11032 Recovery OECD III-1 T=0	838.4	0.0272	27.2
T11032 Recovery OECD III-2 T=0	838.4	0.0266	26.6
T11032 Recovery OECD I-1 T=24	8.0	<LOD	<LOD
T11032 Recovery OECD I-2 T=24	8.0	<LOD	<LOD
T11032 Recovery OECD II-1 T=24	81.9	<LOD	<LOD
T11032 Recovery OECD II-2 T=24	81.9	<LOD	<LOD
T11032 Recovery OECD III-1 T=24	838.4	<LOD	<LOD
T11032 Recovery OECD III-2 T=24	838.4	<LOD	<LOD

The analytical results demonstrate the very low solubility of the test chemical in the test medium and the subsequent rapid degradation after 24 hours representative of the end of the study.  Essentially when considering the factor 10 difference between each of the measured concentrations of the parent material only differed marginally from one another at the start of the test. This indicates that the test chemical was at or close to its maximum achievable solubility limit in test medium and the concentration of parent material was essentially the same regardless of loading concentration. The dose response curve indicates however that additional non quantified components of the test material (most likely to be degradation products) are formed due to the effect on the test organism increasing with loading concentration. This corresponds to the thermal instability known for this test substance.

Validity criteria fulfilled:

yes

Conclusions:

Due to the very high loadings and the relatively constant measurements of parent test material at concentrations very close to the water solubility and bearing in mind the significant number of preliminary tests conducted, the substance can be concluded as non toxic at its solubility limit in the test medium. The effects observed with elevating loading were likely as a result of increasing breakdown product formation formied via hydrolytic and thermal decomposition. Results are such that the test substance and resulting degradation components would not be classified for toxicity to algae.

Executive summary:

GLP report to appropriate guideline. Limitations with analysis due to the instable nature of the test material mean only parent material could be quantified and first analytical method was not sufficiently reliable so was repeated (separate to the test) with a valid method. Reliable with restrictions. Degradation products not quantified.

Description of key information

The substance can be concluded as non toxic at its solubility limit in the test medium.