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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-02-17 to 2012-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed according to current guideline under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the test media of all test concentrations and the solvent control at the start of the test (without algae) and at the end of the test (containing algae). One sample was also taken from the application solution of the highest test concentration at the start of the test.

For the 72 -hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 60 mL) was too small to perform the analyses. A volume of 100 mL per sample was necessary for analytical purposes.

All samples were immediately extracted with ethyl acetate and aliquots of the extracted samples were dried with sodium sulphate. Extracts were stored at about 5°C until analysis.

The concentrations of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide were was determined in the duplicate test medium samples from all test concentrationsgroups. From the solvent control one of the duplicate samples was analyzed per sampling time.
Details on test solutions:
Due to the low solubility and instability of the test item in the test water the organic solvent N,N-dimethylformamide (DMF) was used to dose the test item. The solvent was chosen based on its solubilizing properties and its relative non-toxicity to algae.

The application solution for the dosage of the highest test concentration was prepared by dissolving 83.49 mg of the test item in 10 mL of DMF. This application solution was diluted with DMF to prepare the application solutions for the dosage of the lower test concentrations.

For the preparation of the test media, the volume of 60 µL was added to 1000 mL of test water during intense stirring (10 minutes). For the preparation of the solvent control, the same volume of DMF (without test item) was added to the test water.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000 [2].

The test media were prepared just before the start of the test
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Age of inoculum (at test initiation):
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines. An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Nygaard et al. [1] recommended describing the taxa within the Genus Raphidocelis HINDAK as:

Raphidocelis subcapitata (KORSIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSIKOV
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
15 mg/l as CaCO3 (0.15 mmol/L) - calculated
Test temperature:
21 - 22 °C
pH:
7.5, maintained with 6 mmol/L HEPES buffer
Dissolved oxygen:
not measured
Salinity:
not measured
Nominal and measured concentrations:
Nominal concentrations tested: 31, 63, 125, 250 and 500 µg/L. A solvent control and an untreated control (test water without test item) were tested in parallel. The selection of the test concentrations was based on the results of a pre-experiment to determine the solubility of the test item in the test water and on the results of a range-finding test. In accordance with the test guidelines, test item concentrations significantly above the solubility limit of the test item in the test water were not tested.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml Erlenmeyer flasks
- Type (delete if not applicable): closed (glass stoppered)
- Material, size, headspace, fill volume: fill volume 60 ml to eliminate head space due to potential volatility of the test substance
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): not renewed
- Initial cells density: 10000 cells/mL nominal
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): three
- No. of vessels per vehicle control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS

Ingredients Concentration
Macro-nutrients
NaHCO3 255 mg/L
K2HPO4 1.044 mg/L
MgSO4 × 7 H2O 14.6 mg/L
MgCl2 × 6 H2O 12.16 mg/L
CaCl2 × 2 H2O 4.41 mg/L
NaNO3 25.5 mg/L
Trace elements
H3BO3 186.0 µg/L
MnCl2 × 4 H2O 415.0 µg/L
ZnCl2 3.27 µg/L
CoCl2 × 6 H2O 1.43 µg/L
CuCl2 × 2 H2O 0.012 µg/L
Na2MoO4 × 2 H2O 7.26 µg/L
FeCl3 × 6 H2O 160.0 µg/L
Na2EDTA × 2 H2O 300.0 µg/L

- Source/preparation of dilution water: sterile, purified waster

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not adjusted. HEPES buffer added to test medium to control pH change
- Photoperiod:
- Light intensity and quality: average 6500 Lux (range 5740 - 6870), measured at nine places in the experimental area. The light intensity over the incubation area deviated by less than ±15% from the average light intensity as recommended by the guideline.
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
>= 236 µg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 236 µg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 236 µg/L
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
>= 236 µg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 236 µg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 236 µg/L
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 500 µg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to this test item concentration.
Reported statistics and error estimates:
The algal growth (biomass) in the solvent control was not statistically significantly different from the algal growth (biomass) in the control after 72 hours of test duration (according to Student-t test,  = 0.05, two-sided).
The test item had no statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the highest mean measured test concentration of 236 µg/L (results of Williams t-test, one-sided smaller,  = 0.05, see Table 2 and Table 3).
Validity criteria fulfilled:
yes
Executive summary:

The influence of the test item di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3. The nominal concentrations of the test item were 31, 63, 125, 250 and 500 μg/L. A control and a solvent control group were tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

The measured concentrations of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide in the test media of the nominal test concentrations of 31 to 500 μg/L were between 56 and 76% of the nominal values at the start of the test. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, 26 to 37% of the nominal values were found. In the application solution of the highest test concentration 89% of the nominal value was found.

The biological results were related to the mean measured concentrations of the test item:

 Nominal test concentration (µg/l)  Mean measured concentration (µg/l) Mean measured concentration (% of nominal) 
 31 12   39
 63  25  40
 125  54  43
 250  115  46
 500  236  47

 

The biological results of the study were as follows:

Parameter (0 -72 hour) Growth Rate  Yield

EC10 (μg/L)

>236

>236

EC20 (μg/L)

>236

>236

EC50 (μg/L)

>236

>236

NOEC (µg/L)

>236

>236

Thus, the test item di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide had no toxic effects

on the growth of Pseudokirchneriella subcapitata up to the solubility limit of the test item in the

test water under the conditions of the test.

Description of key information

One OECD 201 study is available with no toxicity observed up to the water solubility limit

Key value for chemical safety assessment

Additional information

The influence of the test item di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3. The nominal concentrations of the test item were 31, 63, 125, 250 and 500 μg/L. A control and a solvent control group were tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

The measured concentrations of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide in the test media of the nominal test concentrations of 31 to 500 μg/L were between 56 and 76% of the nominal values at the start of the test. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, 26 to 37% of the nominal values were found. In the application solution of the highest test concentration 89% of the nominal value was found.

Thus, the test item di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide had no toxic effects on the growth of Pseudokirchneriella subcapitata up to the solubility limit of the test item in the test water under the conditions of the test.