Reaction mass of 1,2,3,4,5,6,7,8-octahydro-5,5-dimethylnaphthalene-2-carbaldehyde and 1,2,3,4,5,6,7,8-octahydro-8,8-dimethyl-2-naphthaldehyde

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

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiry date: 10-08-2018
Storage: Room temperature and protected from light

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

SAMPLE PREPARATION
- Range finding test: Because of the poor solubility in water of the test item an extraction has been performed to extract the soluble components of the test item in water. The test sample has been prepared at 0.1 g/L in culture medium. The solution has been put under stirring for 48 hours at 10 rpm at room temperature. After 3 hours of settling down the supernatant has been used for the test.
- Definitive test: After the range finding test because of the poor solubility in water of the test item an extraction has been performed to extract the soluble components of the test item in water. The test sample has been prepared at 0.1 g/L in culture medium. The solution has been put under stirring for 48 hours at 10 rpm at room temperature. After 3 hours of settling down the supernatant has been used for the test.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The algal suspension in breeding in Eurofins Biolab has been kept in culture medium in sterile 250 mL Erlenmeyer flasks under the following conditions until the beginning of the test:
- Temperature: 23 ± 2°C
- Lighting: lighting greater than 4300 lux
- Stirring: Steady with electrical stirrer

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

± 2 hours

Test temperature:

22.8 - 23.0 °C

pH:

Beginning of the test: 7.90 - 7.92
End of the test: 8.32 - 8.46

Nominal and measured concentrations:

NOMINAL
- Range finding test: 0.01, 0.1, 1, 10, 100 mg/L
- Definitive test: 6.25, 12.5, 25, 50, 100 mg/L
MEASURED (definitive test)
- T=0: 4.98, 10.49, 20.84, 40.75, 72.64 mg/L
- T=24: 4.85, 10.14, 20.45, 38.00, 76.77 mg/L

Details on test conditions:

CULTURE MEDIUM
10 mL of solution 1 and 1 mL of solutions 2, 3 and 4 have been made up to 1000 mL with water.
- Solution 1: 0.75g NH4Cl, 0.9g CaCl2 2H2O, 0.6g MgCl2 6H2O, 0.75g MgSO4 7H2O, 0.08g KH2PO4 in 500 mL deionized water and sterilised by autoclaving (120°C, 15 min).
- Solution 2: 0.032g FeCl3 6H2O, 0.05g Na2EDTA 2H2O in 500 mL deionized water and sterilised by autoclaving (120°C, 15 min).
- Solution 3: 0.0925g H3BO3, 0.207g MnCl2 4H2O, 0.0015g ZnCl2, 0.00075g CoCl2 6H2O, 5E-6g CuCl2 2H2O, 0.0035g Na2MoO4 2H2O in 500 mL deionized water and sterilised by autoclaving (120°C, 15 min).
- Solution 4: 25g NaHCO3 in 500 mL deionized water and sterilised via membrane filtrations (0.22 µm).

TREATMENT
Algal suspension from stock culture (Starting cellular density: 10^4 cell/mL), has been inoculated in the receivers containing the defined concentration of the test sample. Erlenmeyer flasks containing treated organisms and Erlenmeyer flasks containing control organisms, have been kept in suspension by steady stirring with electrical stirrer under continuous light with fluorescent lamp 8000 lux. Number of replications were: 6 for control and 3 for all treated concentrations in range finding and definitive test

OBSERVATIONS
- The test and control samples were incubated for a period of 72 ± 2 h during which the cell density in each test and control solution was measured every 24 h by Burker chamber.
- pH has been measured at the beginning (T=0 h) and at the end of the assay (T= 72 h).
- Temperature of the ambient has been recorded every 24 hours.
- At the end of the test the quality of algae appearance has been observed using a microscope.
- EC50 was evaluated using EPA Probit Analysis Program Version 1.5.

QUALITY CRITERIA
The assay is valid if the following criteria are met:
- the cell concentration in the control cultures should have increased by a factor of at least 16 within three days which corresponds to a specific growth rate of 0.92/day.
- the mean coefficient of variation for section by section (CVss) specific growth rates in the control cultures must not exceed 35%.
- the coefficient of variation of average specific growth rates during the whole period (CVwp) in replicate control cultures must not exceed 7%.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.608 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.971 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test sample is considered stable in assay conditions because the measured concentration of the test item at the end of the test (T=72 h) has been maintained within ± 20 % of the concentration at the beginning of the test (T=0 h).

Validity criteria fulfilled:

yes

Description of key information

The 72h-ErC50 was determined to be 17.608 mg/L.

The 72h-ErC10 was determined to be 6.971 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

17.608 mg/L

EC10 or NOEC for freshwater algae:

6.971 mg/L

Additional information

In an growth inhibition test, performed according to OECD Guideline 201 and following GLP, a culture of Pseudokirchneriella subcapitata algae in the exponential growth phase was exposed to the test item. During the range finding test the algae were exposed to 5 different dilutions (0.01, 0.1, 1, 10, and 100 mg/L) of the test item for 72 hours. After the results of the range finding test an definitive test has been performed. The algae were exposed to 5 dilutions of the test item for 72 hours. The nominal concentrations were 6.25, 12.5, 25, 50, and 100 mg/L and the measured concentrations were 4.98, 10.49, 20.84, 40.75, and 72.64 mg/L and 4.85, 10.14, 20.45, 38.00, and 76.77 mg/L at T=0 and T=72 hours, respectively. Algae have been measured using a Burker chamber every 24 hours. At the end of the test, microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae. The pH in the test vessels was determined to be 7.90 - 7.92 at the beginning of the test and 8.32 - 8.46 at the end of the test. Temperature was recorded every day and ranged from 22.8 - 23.0°C. Stability of the test item in the definitive test has been determined using gas chromatography technique equipped with mass detector and the test sample was considered stable because the measured concentration of the test item at the end of the test has been maintained within ± 20 % of the concentration at the beginning of the test. The ErC50 and ErC10 were determined to be 17.608 and 6.971 mg/L, respectively.