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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 2004-05-09 to 2004-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(1984)
Deviations:
yes
Remarks:
the guideline was slightly modified to ensure a good growth and pH control of the cultures.
Qualifier:
according to guideline
Guideline:
other: EEC (1992): Algal growth inhibition test. Off. J. Of the European communities, L383 A/179, 1992-12-29.
Deviations:
yes
Remarks:
the guideline was slightly modified to ensure a good growth and pH control of the cultures.
Principles of method if other than guideline:
- The NaHCO3 concentration of the test medium was 150 mg/L instead of 50 mg/L, as recommended by the OECD/EEC Guidelines, in order to maintain a more constant pH during the test.
- The pH should not deviate more than 1.5 units during the test (EEC).
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
TEST PRINCIPLE AND PROCEDURE:
For the test an adequate amount of test medium was prepared in a 2 liter vessel. This medium was sterilized by filter sterilization (0.2 µm). Adequate amounts of stock solution were added to the test vessels (100 mL Erlenmeyer flasks). The test vessels were filled with medium up to a total volume of 40 mL using a sterilized dispenser. The test vessels were filled with medium using a sterilized dispenser. Adequate amounts of stock solution were added to the test vessels up to a total volume of 40 mL (100 mL Erlenmeyer flasks). The inoculum was added from an exponentially growing culture with a pipette. In addition six control replicates were included. The extinction in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the spectrophotometer.

PREPARATION OF STOCK SOLUTION:
The test substance is poorly miscible with water. A stock solution of approximately 0.5 g/L of test substance was prepared as follows: to an accurately measured amount of 0.2505 g of test substance 300 mL of OECD medium (pH=7.9) was added. This was transferred to a 500 mL volumetric flask and filled up to the mark with OECD medium. The stock solution was mechanically stirred for approximately 48 hours. Thereafter the stock solution was divided over two separation funnels and left at test temperature for phase separation. After approximately 2.5 hours, when phase separation was completed, the aqueous phase was withdrawn and used to prepare the dilutions. The pH of the aqueous phase was 7.9.

PREPARATION OF 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 organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source: Obtained from the Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, The Windermere Laboratory, Cumbria, Ambleside, United Kingdom.
- Method of cultivation: After purchasing this strain was cultured and maintained according to Standard Operating Procedure E 3 (9.10)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
no data
Test temperature:
22.6 - 23.5 °C
pH:
7.8 - 8.5
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
The test substance is not miscible with water and therefore a 48 hours water accommodated fraction (WAF) was prepared. The green algae were exposed to the following fractions of WAF: 1:32; 1:16; 1:8, 1:4, 1:2 and the undiluted WAF of the test substance in white mineral oil.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks containing 40 mL of medium
- Type: closed
- Initial cells density: approximately 1-10^4 cells/mL
- No. of vessels per concentration: three replicates per test concentration
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: continuously illuminated
- Light intensity: between 95 and 97 µmol/s*m²

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.17 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.88 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.76 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
From the chemical analyses it became clear that the test substance is not stable during the test. This means that due to the followed approach of 48 hours of WAF preparation time, high concentrations of degradation products were present in the WAF and that it is likely that the effects are not caused by the parent compound but by the degradation products. Despite the instability of the parent compound the effects are all calculated based on the concentration of parent compound as measured at the beginning of the test.
Results with reference substance (positive control):
- EC50: between 0.25 and 2.0 mg/L
Validity criteria fulfilled:
yes
Conclusions:
In an algal growth inhibition test with 2,5-dimethyl-2,5-di(t-butylperoxy)hexyne-3 an EC50 (based on biomass) of 6.17 mg/L was determined. The NOEC and LOEC were 1.88 mg/L and 3.76 mg/L, respectively.
Executive summary:

The algal toxicity of 2,5-dimethyl-2,5-di(t-butylperoxy)hexyne-3 was determined in an algal growth inhibition test in accordance with the OECD guideline 201.

The test substance is not miscible with water and therefore a 48 hours water accommodated fraction (WAF) was prepared. The green algae Pseudokirchneriella subcapitata was exposed to the following fractions of the WAF: 1:32, 1:16, 1:8, 1:4, 1:2 and the undiluted WAF of the test item in white mineral oil.

The chemical analyses performed demonstrated a concentration of parent compound in the water accommodated fraction of 3.76 mg/L at the beginning of the test and of < 0.3 mg/L after 24 hours of testing (half-life < 3.4 hours). This means that due to the followed approach of 48 hours of WAF preparation time, high concentrations of degradation products were present in the WAF and that it is likely that the effects are not caused by the parent compound but by the degradation products. Despite the instability of the parent compound, the effects are all calculated based on the concentration of parent compound as measured at the beginning of the test.

The toxicity of the test substance to exponentially growing culture of Pseudokirchneriella subcapitata was determined over an exposure period of 72 hours. The EbC50 (0-72 h) value of the test substance as calculated by extrapolation of the doses effect curve for P. subcapitata is 6.17 mg/L. The NOEC determined from the results is 1.88 mg/L, the LOEC is 3.76 mg/L. The test was conducted in a mineral salts medium in a climatized illuminated orbital incubator. The maximum variation in pH in the test media was 0.7 pH unit.

The definitive test is valid as shown by the increase of the extinction of the control over 72 h by a factor of 124.

Description of key information

In an algal growth inhibition test with 2,5-dimethyl-2,5-di(t-butylperoxy)hexyne-3 an EC50 (based on biomass) of 6.17 mg/L was determined. The NOEC and LOEC were 1.88 mg/L and 3.76 mg/L, respectively.

 

Key value for chemical safety assessment

EC50 for freshwater algae:
6.17 mg/L
EC10 or NOEC for freshwater algae:
1.88 mg/L

Additional information

The algal toxicity of 2,5-dimethyl-2,5-di(t-butylperoxy)hexyne-3 was determined in an algal growth inhibition test in accordance with the OECD guideline 201 (Akzo Nobel 2004).

The test substance is not miscible with water and therefore a 48 hours water accommodated fraction (WAF) was prepared. The green algae Pseudokirchneriella subcapitata was exposed to the following fractions of the WAF: 1:32, 1:16, 1:8, 1:4, 1:2 and the undiluted WAF of the test item in white mineral oil.

The chemical analyses performed demonstrated a concentration of parent compound in the water accommodated fraction of 3.76 mg/L at the beginning of the test and of < 0.3 mg/L after 24 hours of testing (half-life < 3.4 hours). This means that due to the followed approach of 48 hours of WAF preparation time, high concentrations of degradation products were present in the WAF and that it is likely that the effects are not caused by the parent compound but by the degradation products. Despite the instability of the parent compound, the effects are all calculated based on the concentration of parent compound as measured at the beginning of the test.

The toxicity of the test substance to exponentially growing culture of Pseudokirchneriella subcapitata was determined over an exposure period of 72 hours. The EbC50 (0-72 h) value of the test substance as calculated by extrapolation of the doses effect curve for P. subcapitata is 6.17 mg/L. The NOEC determined from the results is 1.88 mg/L, the LOEC is 3.76 mg/L.