Bisisobutyryl peroxide

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:

23-1-2012-27-1-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study to appropriate guideline, with modifications relevant and justified for specific substance characteristics, critical quality criteria were met and endpoints were calculated with cconfidance limits.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Medium adjusted to aid stability tested in sealed system

Principles of method if other than guideline:

Due to the rapid degradation of the test chemical to isobutyric acid (main degradation product) the buffer capacity of the test medium was increased
to assist pH stability. The test chemical was also observed to react with metal components of the test medium (during preliminary studies) resulting innutrient deficiency if not supplemented. 150% concentration of all the standard OECD medium ingredients was therefore used to ensure sufficient
growth.The halflife of the test chemical was such that degradation products were tested as these were considered most relevant for determining
aquatic hazard. A sealed system was used to ensure maximum possible exposure. A WAF (Water Accommodated Fraction) method was used as the
test substance itself is intentionally mixed with iso dodecane for stability / safety reasons and the active component degrades to multiple breakdown
products all of which could potentially contribute to toxicity. Separation and testing of individual components is therefore not possible.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No

Analytical monitoring:

yes

Details on sampling:

10 mL of all test concentrations were sampled for analysis at the start of the stirring period (after 15 minutes of rapid stirring) and at the end of the
stirring period (after 22 hours) and pipetted into sample pots on ice that were subsequently transferred to HPLC vials and analyzed for the active
peroxide component.

Vehicle:

no

Details on test solutions:

Due to the nature of the test chemical being unstable and mixed with iso-dodecane it requires testing in a manner that allows all of its
ingredients or resulting degradation products the possibility to dissolve up to their solubility limit in the test media and thus assessing
ecotoxicological effect of all of the components of the test chemical. For this reason a WAF approach was used for this test. A traditional stock
solution and subsequent dilutions were therefore not made during this test.

A WAF was prepared for each test concentration separately by adding of an accurate amount of the test substance to the test media and allowing
equilibrate under slow agitation over 24 hours in sealed vessels. Each WAF was then considered loaded with the test substance components and / or breakdown products at their corresponding water solubility limits. A GLP Hydrolysis test as well as in test analysis indicates that the main active ingredient of the test substance was no longer present after 24 hours . Erlenmeyer vessels were completely filled with the resulting WAF solutions (essentially degradation products, impurities in iso-dodecane) using a sterile dispenser. Each concentration was tested in triplicate prepared from the same
WAF. The inoculum was then added to the 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.

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.

The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was
prepared to obtain an initial cell density of approximately 100000 cells/mL in each of the test vessels.

Test type:

other: Static Sealed

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Cell obseved for bacterial contamination and morphological abnormalities only.

Hardness:

See other information for medium composition

Test temperature:

The temperature varied from 23.25 to 25.05 °C

pH:

Variation exceeded guideline norms. This is however easily explained by the lower starting pH used to offset a heavy pH decrease due to the acids
formed when the test chemical degrades. 7.1-10.3 in the control indicates good control growth and that the test organism is not effected by a slightly lower start pH.

Dissolved oxygen:

Not measured

Salinity:

See other information for medium composition

Nominal and measured concentrations:

Due to degradation products being tested measured initial concentrations are not relevant for expressing the study endpoints. Nominal or loading
concentrations were therefore used :

4.8, 15.6, 50.0, 160 and 512 mg/L were tested

Details on test conditions:

TEST SYSTEM
- Test vessel: 100ml Erlenmeyer
- Type: closed
- Material, size, headspace, fill volume: Completeley filled no headspace
- Control end cells density:10,000000 cells /ml
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6


GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: in other information

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Highest concentration was tested with and without pH adjustment to demonstrate an actual not pH related effect.
- Photoperiod:Continuous light
- Light intensity and quality:108.0 and 107.6 µmol•m-2•s-1 at the beginning and end of the test respectively


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study : was conducted up to 100 mg/L and from this a definative range exceeding 100 mg/L was chosen due to lack of effect.
- Results used to determine the conditions for the definitive study: Preliminary studies showed removal of Iron from the OECD medium at higher
concentrations. The OECD medium was therefore compensated in the definative test to minimise effects due to unavailable micronutrients.
Previous non GLP research and a GLP hydrolysis study had identified the breakdown products and indicated the short halflife. The test design was
therefore chosen using this information and reccomendations in the "OECD 2000 GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF
DIFFICULT SUBSTANCES AND MIXTURES. (OECD SERIES ON TESTING AND ASSESSMENT NUMBER 23"

Breakdown products include : Isobutyric acid, Isopropanol, propene and acetone

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.5-71.9 mg/L (95% CL)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

151.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 75.1-313.3 (95 % CL)

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

15.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test chemical did cause inhibition of algae growth at high concentrations. Dose response curves with confidence limits were produced. Due to the EL 50 based on growth rate being greater than 100 mg/L the test substance is not classifiable for its toxicity to algae species.

Results with reference substance (positive control):

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.

Reported statistics and error estimates:

The mean values of the observed absorbance for each test substance concencentration were used to calculate the growth and specific growth rate.
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 calculated 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 William’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 compuations were performed using the TOCALC version V 5.0.23 . EC values can also be referred to as EL values when WAF preparations are
used. They are calculated in the same manner. In summary the maximum likelihood probit plot, the Williams test and the Shapiro-Wilks and the
Bartlett’s tests were used.

Medium Composition

Nutrient	Concentration (mg/L)
Macro-nutrients
NH4Cl	22.5
KH2PO4	2.4
CaCl2(H2O)2	27
MgSO4(H2O)7	22.5
MgCl2(H2O)6	18.0
Fe-EDTA
FeCl3(H2O)6	0.096
Na2EDTA(H2O)2	0.15
Trace elements
H3BO3	0.277
ZnCl2	0.0045
MnCl2(H2O)4	0.622
CoCl2(H2O)6	0.0022
CuCl2(H2O)2	0.15x10^-5
Na2MoO4(H2O)2	0.0105
NaHCO3
NaHCO3	150 [Modified]
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid.	200 (HEPES buffer) [Modified]

Results Table

Concentration	ABSORBANCE   Time (hours)				AUC	Inhibition %	SGR	Inhibition %
(mg/L)	0	24	45	72
0.000	0.011	0.059	0.350	0.603	16.392		0.057
0.000	0.009	0.026	0.290	0.574	13.932		0.062
0.000	0.008	0.044	0.274	0.556	13.824		0.061
0.000	0.009	0.032	0.291	0.599	14.400		0.062
0.000	0.007	0.033	0.271	0.619	14.304		0.065
0.000	0.009	0.026	0.303	0.646	15.108		0.064
Mean	0.009	0.037	0.297	0.600	14.660	0.0	0.061	0.0
4.800	0.009	0.043	0.272	0.583	14.016		0.060
4.800	0.010	0.046	0.312	0.675	16.092		0.061
4.800	0.008	0.041	0.266	0.601	14.100		0.062
Mean	0.009	0.043	0.283	0.620	14.736	-0.5	0.061	0.8
15.600	0.008	0.035	0.264	0.664	14.664		0.063
15.600	0.009	0.044	0.261	0.707	15.264		0.061
15.600	0.008	0.040	0.255	0.674	14.688		0.062
Mean	0.008	0.040	0.260	0.682	14.872	-1.4	0.062	-1.1
50.000	0.008	0.042	0.203	0.488	11.256		0.056
50.000	0.008	0.044	0.224	0.582	12.936		0.059
50.000	0.007	0.044	0.223	0.511	12.120		0.058
Mean	0.008	0.043	0.217	0.527	12.104	17.4	0.058	6.0
160.000	0.008	0.029	0.041	0.041	1.692		0.021
160.000	0.008	0.019	0.034	0.043	1.308		0.022
160.000	0.008	0.025	0.035	0.038	1.416		0.020
Mean	0.008	0.024	0.037	0.041	1.472	90.0	0.021	65.8
512.000	0.002	0.015	0.021	0.022	1.008		0.017
512.000	0.004	0.010	0.021	0.020	0.744		0.014
512.000	0.003	0.015	0.027	0.028	1.164		0.019
Mean	0.003	0.013	0.023	0.023	0.972	93.4	0.017	72.9

AUC= Area under curve (Biomass)

SGR = Specific Growth Rate (Rate)

Validity criteria fulfilled:

yes

Conclusions:

The study can be considered reliable without significant restriction.

Executive summary:

A guideline study to GLP with supplementary chemical analysis and satisfactory substance identification was carried out with justified deviations from the guideline due to the inherent characteristics of the tests substance. All critical validity criteria were met and the study can be considered a reliable worst case representation of the effects the test substance to P.Subcapitata. Due to the relatively low toxicity observed (EL50 >100 mg/L) and the ready biodegradability of the test substance and it breakdown products the fact that the breakdown products were not separately quantified is not considered a critical flaw to the study.

Description of key information

Due to the extremely short half-life of the test material determined in the hydrolysis study the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. All test concentrations were prepared separately as Water Accommodated Fractions to allow the determination of the toxicity of the resulting degradation mixture as a whole to be determined. It is known that the test chemical degrades to multiple degradation products and is itself present in isododecane solvent. The effects cannot therefore be attributed to a single component with 100% certainty however the main degradation product isobutryic acid is the most likely cause of the slight toxicity observed.  

ECHA data on Isobutyric acid gives 72h-EC50 values of 45.1 mg/L (biomass) and 44.7 mg/L (fluorescence) (ECHA dossier 79 -31 -2)

.

Key value for chemical safety assessment

EC50 for freshwater algae:

151.2 mg/L

EC10 or NOEC for freshwater algae:

35 mg/L

Additional information

Due to the extremely short half-life of the test material determined in the hydrolysis study the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. Effects to algae were observed and an EL10 and an EL50 were determined. The test material is not of high concern to algae species as the EL50 was greater than 100 mg/L of test material and would therefore not be classified for this endpoint. For the algae endpoint exact EL50 values were determined. For this reason the algae endpoint was used for PNEC derivation.