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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
corrected 28 July, 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
p-tert-butylaniline
EC Number:
212-215-9
EC Name:
p-tert-butylaniline
Cas Number:
769-92-6
Molecular formula:
C10H15N
IUPAC Name:
p-tert-butylaniline

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.75, 1.73, 3.97, 9.13 and 20.99 mg test item/L
- Sampling method: Samples (2 x 7.5 mL) were taken from all five test solutions and controls at the beginning of the test prior to addition of the algae (2 x 6 samples). At the end of the growth test, samples were taken directly from representative replicates per test concentration and control (2 x 6 samples). Additionally, samples after 24 and 48 h were taken but not analyzed, since the results obtained at test start and test end were sufficient for the evaluation of the study.
A volume of 7.5 mL of sample was used and all samples were stabilized with 7.5 mL of acetonitrile immediately after the sampling.
- Sample storage conditions before analysis: Samples were stored at -20°C in a refrigerator until shipment to the partner lab for analysis. For security reasons, a reserve set of stabilized samples was stored deep frozen.

Test solutions

Vehicle:
no
Remarks:
medium was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The sterilised synthetic growth medium (OECD medium) according to OECD 201 was used as growth medium.
All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. The pH of the medium was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.
The highest test concentration was prepared by weighing in 21.15 mg of test item and transferring it into a glass bottle. Afterwards, 1 L of growth medium was added and the treatment was stirred for about 24 hours at room temperature. The other test concentrations were prepared by serial dilution of the highest test concentration with growth medium using a spacing factor of around 2.3.
All work was conducted on a clean bench using sterile equipment.

Before test start, an appropriate stock solution was prepared by weighing in 1.5 mg of test item and transferring it to 1L Cu-reduced dilution water. The stock solution also served as highest test concentration. The stock solution was stirred for about 24 hours at room temperature.
The other test concentrations were prepared by dilution of the stock solution with Cu-reduced dilution water using a spacing factor of 2.2.
- Controls: growth medium without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially growing algae for the test. The culture duration of the pre-cultures was 3 days.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
21 to 24 °C, controlled at ± 2°C
pH:
7.3 - 7.54
Nominal and measured concentrations:
Nominal: 0.75, 1.73, 3.97, 9.13 and 20.99 mg test item/L
Measured: 0.646, 1.45, 3.45, 8.40 and 19.05 mg test item/L
Since the concentration of the test item were maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, analysis of the results presented was based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Test vessels were 250 mL conical glass flasks covered with air-permeable silicone-sponge caps. The vessels and caps were sterilized prior to use (autoclaving).
- Type (delete if not applicable): closed
- Renewal rate of test solution (frequency/flow rate): The main test was conducted as static test without renewal of test medium.
- Initial cells density: 1.828 x 106 cells/mL
- No. of organisms per vessel: The algal pre-culture (549.5 µL at a cell density of 1.828 x 106 cells/mL) was added to the test vessels to achieve the initial cell concentration of 10,000 cells/mL.
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes (OECD medium of OECD TG 201 was used)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: light intensity adjusted to 60 - 120 µE m-2 s-1 (4440 - 8880 lux) close to the surface of the liquid. The light intensity was measured daily using a cosine (2 π) receptor in µE m-2 s-1 at level of the test media surface.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.
- Determination of cell concentrations: Cell concentrations were determined using an electronic particle counter (CASY 1 Model TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany). During the test, the cell concentrations were determined after 24, 48 and 72 h in samples taken directly from the test vessels.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2.3
- Range finding study: A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test.
- Test concentrations: 12.5, 25, 50 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.87 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
9.13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.97 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.04 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.97 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.73 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Results with reference substance (positive control):
The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest (December 2017) ErC50 value of 2.90 mg/L (nominal; 95% confidence limits: 2.66 – 3.16 mg/L) for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD TG 201 without deviations, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable:
There were concentration-dependent inhibiting effects on the growth of the freshwater green algae observed over the range of the tested concentrations. The respective 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated to be 2.98 mg test item/L and 8.87 mg test item/L. For yield, an EyC10 and EyC50 of 3.31 and 5.04 mg test item/L were determined, respectively.
The NOEC for growth rate was set to be at 3.97 mg test item/L, whereas a NOEC of 1.73 mg test item/L was determined for the inhibition of yield.
Based on these results (related to the inhibition of growth rate), the test item has to be classified as chronic Cat. 2 (substance is soluble in water and not readily biodegradable), but the actual classification is based on the most sensitive species (daphnia magna).
Executive summary:

A GLP study was performed to determine the toxicity of the test item p.-tert. Butylanilin on the growth of the unicellular freshwater green alga Raphidocelis subcapitata.

Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

 The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations: Control, 0.75, 1.73, 3.97, 9.13 and 20.99 mg test item/L.

The concentrations of the test item in the media were confirmed by measurements of the test item concentrations before and after the test by HPLC-UV/VIS (LOQ = 0.471 mg/L).

At test initiation, the measured concentrations were 0.752, 1.74, 4.03, 9.38 and 21.6 mg test item/L (100.3 – 103.0 % of nominal).

After 72 hours, the measured test item concentrations were 0.646, 1.45, 3.45, 8.40 and 19.05 mg test item/L (83.5 – 91.9 % of nominal; 83.2 – 89.5 % of initial).

Since the concentration of the test item were maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, the results presented are based on nominal concentrations.

There were concentration-dependent inhibiting effects on the growth of the freshwater green algae observed over the range of the tested concentrations.

The respective 72-hour ErC10 and ErC50 for the inhibition of growth rate were estimated to be 2.98 mg test item/L and 8.87 mg test item/L.

For yield, an EyC10 and EyC50 of 3.31 and 5.04 mg test item/L were determined, respectively.

The NOEC for growth rate was set to be at 3.97 mg test item/L, whereas a NOEC of 1.73 mg test item/L was determined for the inhibition of yield.