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Diss Factsheets

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
Study period:
19.11.2012 to 22.11.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2011)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Schmallenberg (Germany)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrabutylurea
EC Number:
224-929-8
EC Name:
Tetrabutylurea
Cas Number:
4559-86-8
Molecular formula:
C17H36N2O
IUPAC Name:
1,1,3,3-tetrabutylurea
Details on test material:
- Name of test material (as cited in study report): Tetrabutyl urea; 1,1,3,3-tetrabutylurea
- Substance type: amide
- Physical state: liquid
- Analytical purity:99.6%
- Impurities (identity and concentrations): dibutylamine, water, chloride, sulfur
- Composition of test material, percentage of components: dibutylamine: 0.04%, water: 0.04%, chloride: 0.8 ppm, sulfur:<1 ppm
- Purity test date: 04.08.2012
- Lot/batch No.: 2011-11-4
- Expiration date of the lot/batch: 03.11.2013
- Storage condition of test material: test substance stored dry at room temperature (15-25°C) in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of tetrabutyl urea was analysed in all test solutions sampled at the beginning of the exposure period prior to the addition of the algae and in samples of the algae cultures taken from replicates pooled per test concentration at the end of the exposure period. Duplicate samples of approximately 20 mL were taken. The chemical analysis was performed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The samples were analysed immediately on the sampling day. Spare samples were frozen immediately and stored at -20°C.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution and the highest test concentration were prepared by dissolving 4.3 mg of the test item in 1000 mL growth medium and stirring vigorously for about 15 min at room temperature. The other four test concentrations of nominal 2.15, 1.075, 0.538 and 0.269 mg/L were obtained by dilution of the stock solution.
- Controls: Flask filled with growth medium served as control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Göttingen (Germany)
- Age of inoculum (at test initiation): exponentially-growing algae (not further specified)
- Method of cultivation:The stock cultures are maintained fulfilling the criteria of the OECD Guideline 201. Prior to testing, a pre-culture is established in standard OECD growth medium to obtain exponentially-growing algae for the test.

ACCLIMATION
- Acclimation period: not necessary
- Culturing media and conditions same as test: yes
- Any deformed or abnormal cells observed: no

Study design

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

Test conditions

Test temperature:
21.0-21.5°C
pH:
controls
test start: 8.14
test end: 8.05-8.44

test vessels
test start: 7.86-8.34
test end: 8.01-8.48
Nominal and measured concentrations:
nominal: 0 (control), 0.269, 0.538, 1.075, 2.15, and 4.3 mg/L
geometric mean measured: 0 (control), 0.05, 0.14, 0.58, 1.56, and 2.95 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): the test vessels were covered with air-permeable silicone-sponge caps
- Material, size, headspace, fill volume: glass/250 mL/150 mL/100 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Renewal rate of test solution (frequency/flow rate): test conducted under static conditions
- Initial cells density: 10000 cells/mL
- Control end cells density: 1082000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature was measured daily, pH on day 0 and after 3 d

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7518-7578 lux (OSRAM "cool white")

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth rate, yield
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: performed
- Test concentrations: 0.1, 1.0, and 10 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.56 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.95 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
(yield)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.58 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
(yield)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
(yield)
Remarks on result:
other: 95% c.i.: 0.85-1.42 mg/L
Results with reference substance (positive control):
Results with 3,5-dichlorophenol:
ErC50=2.42 mg/L nominal (95% confidence limits: 1.95-3.03 mg/L)
These results are in good agreement with the results of an international ring test with ErC50=3.38 +/- 1.30 mg/L
Reported statistics and error estimates:
Probit-analysis was used for the determination of EC50, EC20 and EC10 values together with 95% confidence intervals in respect to yield. The NOEC and LOEC values for growth rate and yield were determined using the Williams t-test. The computer program ToxRat Professional was used for all statistical evaluations.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of tetrabutyl urea to the unicellular, green alga Pseudokirchneriella subcapitata was investigated in a study conducted according to OECD Guideline 201. After 72 h of exposure, NOEC values of 0.14 mg/L and 0.58 mg/L were determined for yield and growth rate inhibition, respectively. The 72 h EC50 for yield was determined to be 1.11 mg/L. Since the growth rate inhibition was below 50 % compared to controls at the highest test concentration the 72 h EC50 value was determined to be higher as the highest test concentration (72 h EC50 > 2.95 mg/L; all effect concentrations based on geometric mean measured concentrations).
Executive summary:

The toxicity of tetrabutyl urea to the unicellular, green alga Pseudokirchneriella subcapitata was investigated in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test; 2011). In this static test, exponentially growing cultures of the alga were exposed to nominal tetrabutyl urea concentrations of 0 (control), 0.269, 0.538, 1.075, 2.15, and 4.3 mg/L for 72 h. The cell density in each test culture was measured at least every 24 hours. The inhibition of growth in relation to the control cultures was determined for every exposure period. The concentration of tetrabutyl urea was assessed by chemical analysis (LC-MS/MS) at the beginning and at the end of the test. As the measured tetrabutyl urea concentrations were outside of the ± 20 % range of the measured initial concentrations for some treatment levels at the end of the study, the geometric mean measured concentrations for each test concentration was calculated resulting in actual concentrations of 0.05, 0.14, 0.58, 1.56, 2.95 mg/L. After 72 h exposure, NOEC values of 0.14 mg/L and 0.58 mg/L were determined for yield and growth rate inhibition, respectively. The 72 h EC50 for yield was determined to be 1.11 mg/L. Since the growth rate inhibition was below 50 % compared to controls at the highest test concentration the 72 h EC50 value was determined to be higher as the highest test concentration (72 h EC50 > 2.95 mg/L; all effect concentrations based on geometric mean measured concentrations).

The study is regarded as reliable without restriction and fulfills the requirements of the guideline.

Results Synopsis

Test organism: Pseudokirchneriella subcapitata

Test type: static

growth rate

72 h NOEC=0.58 mg/L

72 h LOEC=1.56 mg/L

72 h EC50>2.95 mg/L

yield

72 h NOEC=0.14 mg/L

72 h LOEC=0.58 mg/L

72 h EC50=1.11 mg/L

based on geometric mean measured concentrations

Endpoint(s) effected: growth rate, yield