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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
72 h
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Details on test solutions:
The stock solution was prepared by measuring 80.32 mg of test item into 1 000 ml OECD growth medium. The suspension was stirred and ultrasonicated till complete dilution. The measured concentration of stock solution was 79.53 mg/L.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test species: Pseudokirchneriella subcapitata (Korshikov) F.Hindák (formerly known as Selenastrum capricornutum and Raphidocelis subcapitata)
Source: Culture Collection of Algae and Protozoa, Scottish Marine
Institute (www.ccap.ac.uk)
Batch: CCAP 278/4
Date of arriving: 27 October 2017

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
The temperature during the test remained in the 21 ± 2 °C range. The temperature data were harvested by Extech SD200 datalogger.
The temperature inside the incubator at the test vessels was measured and registered continuously by Extech SD200 thermometer and datalogger. Throughout the test the temperature was nearly constant.
pH:
At the beginning of the test the pH values were measured in the remaining test solution of the concurrent test vessels at each dilution level. At the end of the test the pH values were measured in each test vessel. The pH of test solutions was not adjusted at the beginning of the test.
Nominal and measured concentrations:
The toxic property of test item was investigated at seven concentrations which were
prepared by different dilutions of stock solution of test item. OECD medium was used
for dilution of stock solution. In the test the following calculated concentrations were
applied: 79.53; 31.81; 15.91; 7.95; 3.98; 1.59 and 0.80 mg/L.
Details on test conditions:
Preparation and breeding of inoculum culture: The inoculum culture was prepared four days before starting the test and it was bred in incubator at 21 ± 2 °C. At the end of incubation the cell density of the inoculum culture was determined with a Bürker chamber. The cell concentration was 1 930 556 cell/ml
Preparation of stock solution: The preparation of stock and test solutions was based on the preliminary non GLP range finding test [9]. As the Range Finding Test was not performed in compliance with the GLP-Regulations it is excluded from the Statement of Compliance in the final report, but the raw data of this test are archived under the study code of present study. The stock solution was prepared by measuring 80.32 mg of test item into 1 000 ml OECD growth medium. The suspension was stirred and ultrasonicated till complete dilution. The measured concentration of the stock solution was 79.53 mg/L.
Preparation of test solutions: Seven test solutions of different concentration were prepared by mixing appropriate volume of OECD medium and stock solution of the test item. The maximum separation factor between of test vessels was 2.5. Two additional replicates were prepared for controlling the abiotic degradation of the test substance. These test vessels were not inoculated with alga inoculum culture. The following table shows the preparation of test solutions. The volume of the prepared dilution levels was 300 ml except the control solution which was 600 ml. The alga inoculum culture was diluted 389.61 times so the initial alga cell concentration in every dilution and the control levels were 4 955 cells/ml. 5.4. Preparation of test vessels Three replicates were prepared at every test, two at the abiotic control and six at the control level. The vessels contained 75 ml test media.
Test procedure: The test vessels were closed with silicone stopper and kept in the Binder KBW 400 incubator. For the proper mass transfer of carbon dioxide glass tubes with filter were placed through silicone stoppers. The test was maintained under static conditions for a period of 72 hours. The temperature during the test remained in the 21 ± 2 °C range. The temperature data were harvested by Extech SD200 datalogger. The vessels were shaken and illuminated continuously in the incubator. The vessels were placed randomly into the incubator and were repositioned daily after sampling. One of the vessels of abiotic controls was taken into the incubator with a complete aluminium foil cover to check the stability of test item in the absence of light. This vessel was signed as 8/B (vessel No: 23). The abiotic control on light were signed as 8/A (vessel No: 22).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
7.61 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The average specific growth rate is a response variable which is calculated on the basis of the logarithmic increase of alga biomass during the test, expressed per day. The effective concentrations for growth rate are signed as ErCx. The ErCx values were determined including the 95 % confidence interval with Probit analysis using linear max. Likelihood regression. The ErC10, ErC20 and ErC50 were calculated for at both 24, 48 and 72 h period.
The No Observed Effect Concentration and Lowest Observed Effect Concentration for the test item were calculated by ToxRat® software for specific growth rate and yield. The results were not determinable.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Acute toxic effect of 15α-hydroxy-androstenedione was investigated in Freshwater Alga and Cyanobacteria, Growth Inhibition Test (according to OECD 201 guideline). The ErC50 was 13.67 mg/L at 72 h. According to the Council Regulation (EC) 440/2008 15α-hydroxy-androstenedione is hazardous to the aquatic environment and classified into the second acute toxic hazardous category [GHS Acute aquatic hazard Category Acute III. – 72 h EC50 (for algae or other aquatic plants) > 10 mg/L but ≤ 100 mg/L].