Dioctanoyl 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:

Experimental starting date 3-11-2015 Experimental completion date 6-11-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Deviations from the guideline
• Increased levels of NaHCO3 (150 mg/L) will be added to the test mediu m to assist with pH stabilization this differs with the guideline recommended concentration of 50 mg/L.
• The solubility limit of the test substance is below the limit of quantification of the analytical method and therefore analysis could not be conducted.
• Due to the poor solubility and the fact that solubility should not be exceeded a WAF (Water Accommodated Fraction) method was used to test at the water solubility limit of the parent
material as well as any water soluble impurities that may be present or any degradation products that may have accu mulated during the stirring period.
• A stable dissolved concentration in the WAF solutions could not be demonstrated due to lack of analysis. For this reason extended stirring periods of 1 -7 days were tested during preliminary testing to establish if an increase in toxicity took place (indicating increased test substance concentration) and allow the worst case to be tested during GLP studies. A 7 day stirring period was therefore chosen during this test as this was the only preliminary study in which inhibition to the test organism was observed.
• Due to extended stir iron precipitation in the test mediu m may be observed. In such cases 50% of the original concentration of chelated iron (media component) was added to the WAF prior to testing to prevent shortage of this essential micronutrient.
• Test concentrations did not have an even spacing between them as is recommended in the test guideline. The highest concentration should have ideally been 160 mg/L. This did not affect the ability to calculate the required endpoints over the range of concentrations tested. The EC50 may still be concluded as > the highest concentration.

Deviations:

yes

Remarks:

see version/remarks

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Solubility: <10 μg/L
Storage: -20°C
Batch/Lot: 1204350050

Analytical monitoring:

no

Details on sampling:

The solubility of the test substance was below the detection limit of the available analytical method.
Analysis was therefore not possible.

Vehicle:

no

Details on test solutions:

Test medium
The test medium (OECD algae medium) was prepared according to the OECD and EC guideline (OECD, 2011 and Commission Regulation, 2008) with the following modification: The NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain a more constant pH.
The test medium was prepared from concentrated solutions of the mineral salts prepared in deionized water and filter sterilized.

Preparation of the test solutions
Due to the low solubility of the test substance preparation of the test solutions in a standard manner via a stock solution and further dilution was not possible. For this reason WAF's were prepared for each test concentration separately. This was achieved by accurate weighing of the test substance with an analytical balance and loading of the test material to 1 liter of test medium. The resulting solutions were stirred slowly for an extended period 7 days to ensure maximum solubility had been reached. This time span was shown in preliminary studies to cause some slight effects in algae species (compared to shorter stirring periods) and was therefore in the absence of analysis presumed to be the worst case scenario. After 7 days of slow stirring the solutions were stopped and allowed to rest for one hour. After which liquid from each of the WAF vessels was transferred directly to the test vessels using a calibrated dispenser pump extracting liquid from the middle of the WAF flask to ensure that only the soluble fraction was transferred. In order to minimize possible loss of the substance to glass all vessels were filled then emptied and refilled again in the same manner. Additional iron was then added to each vessel to compensate for the iron that had precipitated out of solution during the stirring period. The test solutions were then ready for addition of the test organisms and for physical chemical/absorbance measurements. Finally all vessels were incubated for 72 hours with absorbance measurements made every 24 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 (CCA P 278/4) obtained from the Culture Collection of Algae and Protozoa SAMS Research Services Ltd. Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4 °C in the dark 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.
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, and is not used unless found to be between the set EC50 values of 0.25 to 2.0 mg/L as indicated in the study guideline.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

23 ± 2°C

pH:

7.5-7.8

Conductivity:

< 5 μS/m

Nominal and measured concentrations:

Nominal concentrations: 10, 20, 40, 80, 100 mg/L loading rate.

Details on test conditions:

De-ionised water
The de-ionised water used in the study contained less than 10 μg/L of copper (not measured under GLP), with a conductivity of less than 5 μS/m and less than 2.0 mg/L NPOC-content.

Test vessels
The test was performed in 100 ml Erlenmeyer flasks containing 40 ml of medium. The test flasks were closed with cotton-wool stoppers.

Culturing cabinet and test conditions
The test was carried out in a temperature-controlled illuminated orbital incubator, in which the
temperature was maintained at 23 ± 2°C, and a continuous uniform illumination was provided in the
spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36 ± 0.02 m from the algal cultures. The light intensity was in the range of 60 to 120 μ E· m-2·s-1 (= μmol.m-2·s-1). The test vessels were rotated continuously at a speed sufficient to prevent sedimentation of the algae.

Test conditions
Based on the results of a series of range finding studies some slight inhibition occurred at 100 mg/L when an extended stirring period was used. The following final nominal test substance loadings were prepared:
10, 20, 40, 80 and 100 mg/L.

Preparation of the inoculum
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 extinction of an exponentially growing stock culture was measured. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1·10^4 cells/ml in the test medium.

Test procedures
Culture medium was prepared by diluting the stock mineral salts in an appropriate vessel. This medium was sterilized by filter sterilization (0.2 μm). Adequate a mounts of test substance stock solution were added. The Erlenmeyer flasks were then filled with test solution up to a total volume of 40 ml using a sterilized dispenser pipette. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control were included.
The extinction of light in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Algal
medium was used as a blank in the spectrophotometer.

Determination of cell concentrations
Cell concentrations were determined photo metrically with a UV/VIS Spectrophotometer. Measurements were carried out at 436 n m in a cuvette with a light path of 4 cm. To establish the relation between extinction of light and cell density, a calibration curve was made which is checked yearly. From the relation between extinction (E) and counted cell number per ml (N) the following calibration curve was determined using linear regression:
N = (E-0.0386)/4.10^-7
The calibration curve was used to determine the cell density of the inoculum before and during the test when needed.

Evaluation of data
The raw data was evaluated using the Toxrat V2.10 statistical software package using the template for the appropriate study type. Toxrat V 2.10 is a tailor made program for the evaluation of toxicity studies.
The selection of the most appropriate statistical tests is carried out by the software package. A complete report has been included in the raw data and extracts relevant to the test guideline requirements have been reported.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Remarks:

ErL10

Effect conc.:

90.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

ErL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Remarks:

EyL10

Effect conc.:

36.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Remarks:

EyL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Significant effect was not observed on growth rate. The NOELR (growth rate) is therefore ≥100 mg/L and LOELR was not determined. The ErL10 was determined as 90.5 mg/L. The ErL50 was > 100 mg/L.
Significant effect was observed on yield. The NOELR (yield) is therefore 80.0 mg/L and LOELR was 100.0 mg/L. The EyL10 was 36.7 mg/L. The EyL50 was > 100 mg/L.

Results with reference substance (positive control):

The following quality criteria have been met in the present study:
• The cell density of the controls increased at least a factor 16 within 72 hours.
• The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.
• The coefficient of variation of average specific growth rates during the whole test period in the
replicate control cultures should not exceed 7%.
• Algae strain used was of acceptable sensitivity according to a recent reference test.

The pH measurements show a maximum increase of 1.6 pH units that is slightly higher than the 1.5 indicated in the test guideline. This is not a quality criterion and is not considered to

affect the studies reliability.

The temperature varied fro m 23. 1 to 23.6 ° C during the test.

Light intensity was 100.4 and 99.6 μmol·m^-2·s^-1 at the beginning and end of the test, respectively.

All measurements are in accordance with the required conditions described in the study plan.

Five random samples of the control were all pure and not contaminated with bacteria.

Test parameters and summary of the results based on nominal loadings

 

Parameter	Name	Dimension	Value
-	Increase factor of cell growth over 72 h	-	202
µ	Growth rate (control)	d-1	1.7
NOEC	No observed effect concentration (rate)	mg/L	≥100
LOEC	Lowest observed effect concentration (rate)	mg/L	Not determined
		Value (mg/L)	95% confidence limits
EyC10		36.7	-	-
EyC20		55.9	-	-
EyC50		124.8	-	-
ErC10		90.5	84.5	96.4
ErC20		103.7	97.2	116.0
ErC50		134.4	119.0	176.2

- Not Calculated

Ey - Effect Yield

Er - Effect Specific Growth rate

pH-values

Actual test substance loadings (mg/L)	Time (hours)
	0*	72
0	7.8	9.3
	7.8	9.4
	7.8	9.2
	7.8	9.3
	7.8	8.4
	7.8	9.2
Rep	7.8	-
10	7.6	8.2
	7.7	8.4
	7.7	8.3
Rep	7.6	-
20	7.7	9.0
	7.7	9.1
	7.7	9.2
Rep	7.7	-
40	7.7	9.1
	7.7	9.0
	7.7	9.2
Rep	7.7	-
80	7.7	9.0
	7.8	9.0
	7.8	9.0
Rep	7.8	-
100	7.5*	8.1
	7.5*	8.2
	7.5*	8.3
Rep	7.5	-

*Adjusted to pH with 0.1 NaOH

Validity criteria fulfilled:

yes

Conclusions:

Significant effect was not observed on growth rate. The NOELR (growth rate) is therefore ≥100 mg/L and LOELR was not determined. The ErL10was determined as 90.5 mg/L. The ErL50was estimated as >100 mg/L.
Significant effect was observed on yield. The NOELR (yield) is therefore 80.0 mg/L and LOELR was 100.0 mg/L. The EyL10was 36.7 mg/L. The EyL50was > 100 mg/L.

Executive summary:

In order to estimate the effects of the test chemical in an aquatic environment, the toxicity of the soluble components of the test substance to freshwater algae was determined using the Algal Growth Inhibition Test in accordance with OECD, EC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. Slight modifications to the guideline were applied to ensure good growth and pH control of the cultures and appropriate testing of a very poorly soluble substance.

The toxicity of the test substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. Loadings of 10, 20, 40, 80, and 100 mg/L were tested including the required control group.

Due to the poor solubility and the fact that solubility should not be exceeded a WAF (Water Accommodated Fraction) method was used to test at the water solubility limit of the parent material as well as any water soluble impurities that may be present or any degradation products that may have accumulated during the stirring period.

Significant effect was not observed on growth rate. The NOELR (growth rate) is therefore ≥100 mg/L and LOELR was not determined. The E_rL₁₀was determined as 90.5 mg/L. The E_rL₅₀was estimated as >100 mg/L.

Significant effect was observed on yield. The NOELR (yield) is therefore 80.0 mg/L and LOELR was 100.0 mg/L. The E_yL₁₀was 36.7 mg/L. The E_yL₅₀was > 100 mg/L.

The test is valid as shown by:

The cell density of the control increased over 72 h by a factor of 202.

The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.

The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%.

The most recent reference test for the algae species tested demonstrated acceptable sensitivity according to the test guideline.

All of the biological quality criteria were met and the study data can be considered to be an accurate representation of the effects of a water accommodated fraction generated from the nominal loadings of the test material to P.subcapitata.

Description of key information

Significant effect was not observed on growth rate. The NOELR (growth rate) is therefore ≥100 mg/L and LOELR was not determined. The ErL10was determined as 90.5 mg/L. The ErL50was estimated as >100 mg/L.

Significant effect was observed on yield. The NOELR (yield) is therefore 80.0 mg/L and LOELR was 100.0 mg/L. The EyL10was 36.7 mg/L. The EyL50was > 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

90.5 mg/L