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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-11 to 2018-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
corrected version from 2011
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(EC) No. 266/2016
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P1110914CA
- Expiration date of the lot/batch: 2019-02-21
- Purity test date: 2018-02-21

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 18 – 25 °C, dark, in tightly closed original container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Storage at 18 – 25 °C, dark, in tightly closed original container
Analytical monitoring:
yes
Details on sampling:
- Concentrations [mg/L]: 0.158, 0.500, 1.58, 5.00, 15.8, 50.0 (nominal). Additionally the highest test concentration of nominal 50.0 mg/L was tested with pH adjustment to pH 8.1 ± 0.2 by adding 1 M HCl.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

A test item solution with a nominal concentration of 50.0 mg/L was freshly prepared with dilution water one day prior to the start of exposure. The test item was pipetted onto the surface of the dilution water. For this purpose, the density was taken into account. The test item solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. After a separation phase of 3 hours the test item solution was removed by siphoning from the middle of the flask and was used for testing. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. The check revealed no presence of undissolved test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 5143 – 6535 lux for 24 hours per day.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LÜTTGE et al. (1994)
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
The dilution water (OECD TG 201 medium) has a nominal hardness of 0.24 mmol Ca+Mg/L.
Test temperature:
23 - 24 °C
pH:
The pH-value at the start of the exposure and at the end of the exposure were measured from pooled samples of each test item concentration and the control after measurement of the cell density with a pH-Meter pH 191 (WTW).

Range of pH-values for test solutions; start; 0 hours: 8.12-9.04; end, 72h: 8.11-9.33
pH values for control; start, 0 hours: 8.26; end, 72h: 9.51
Nominal and measured concentrations:
The concentrations of the test item Tri-n-butylamin were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) in all concentration levels and in the control.
The measured concentrations of the test item were between 88 and 120% of the nominal value at the start of the exposure (0 hours) and between 79 and 100% of the nominal value at the end of the exposure (72 hours). Since the measured test item concentrations were not within the range of ± 20% of the nominal concentrations, the geometric mean measured concentrations were calculated.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Test volume: 100 mL
- Initial cells density: 6,442 cells/mL
- Control end cells density: Mean value of cell density of all six replicates after 72 hours: 1,833,312 cells/mL
- No. of vessels per concentration (replicates): Three replicates per concentration. Two replicates were tested for the highest test concentration with pH adjustment.
- No. of vessels per control (replicates): Six replicates for the control.

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium according to guideline

TEST MEDIUM / WATER PARAMETERS
A four days old preculture, prepared in dilution water, was used as inoculum.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The highest test concentration of nominal 50.0 mg/L was tested twice, one set of repliceates with pH adjustment to pH 8.1 ± 0.2 by adding 1 M HCl and one without .
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1.
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the concentration 50.0 mg/L.
- Chlorophyll measurement: The fluorescence values were related to cell density values according to a calibration curve.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:√10 = 3.16
- Range finding study: A non-GLP preliminary range finding test under static conditions over a period of 72 hours was conducted at the test facility with three concentrations of the test item with nominal concentrations of 0.500, 5.00, 50.0 mg/L. In the range finding test, two replicates per concentration and four for the control were tested.
- Test concentrations: Six concentration levels in a geometrical series with a dilution factor of √10 were tested: 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L. Additionally the highest test concentration of nominal 50.0 mg/L was tested with pH adjustment to pH 8.1 ± 0.2 by adding 1 M HCl.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate was used as reference item.
Key result
Duration:
72 h
Dose descriptor:
EC10
Remarks:
ErC10
Effect conc.:
4.64 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Remarks:
ErC20
Effect conc.:
6.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Remarks:
ErC50
Effect conc.:
10.1 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.:
1.65 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.:
5.05 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Remarks:
EyC10
Effect conc.:
< 0.154 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Remarks:
EyC20
Effect conc.:
< 0.154 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.154 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Remarks:
EyC50
Effect conc.:
5.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.511 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-03-26 to 2018-03-29. The reference item toxicity is in the valid range following test facility SOPS.
- Growth Rate Inhibition, ErC50: 1.09 compared to valid range of 0.759 ± 0.555
- Yield Inhibition, EyC50: 0.548 compared to valid range of 0.401 ± 0.297
Reported statistics and error estimates:
Please see IUCLID section "Overall remarks, attachments" for details on statisitcal evaluation!

NOEC, LOEC, EC-values and 95% Confidence Intervals of Tri-n-butylamine (0 -72 hours)

 

 Growth Rate Inhibition

Geometric mean test item concentration [mg/L]

 NOEC  1.65
 LOEC  5.05
 ErC10  4.64 (3.97 -5.53)
  ErC20 6.45 (5.74 -7.28) 
  ErC50  10.1 (9.38 -10.9)

 

 

 Yield Inhibition

Geometric mean test item concentration [mg/L]

 NOEC  0.154
 LOEC  0.511
 EyC10  < 0.154
  EyC20 < 0.154
  EyC50 5.13 (4.14 -7.26)

 

Measured Concentrations and Percentage of the Nominal Concentration of Tri-n-butylamine in Fresh Medium (0 hours) and Old Medium (72 hours) with Algae

 Sampling date

    Fresh medium

0 hours

    Old medium

72 hours

 

    Nominal concentration of the test item [mg/L]

          Tri-n-butylamine

Geometric mean concentration [mg/L]

 Meas. conc.[mg/L]

[ %]   Meas. conc. [mg/L] [%] 
 50.0 (pH-adjusted) 45.3 91  45.0  90   
 50.0 43.8  88  42.5  85  43.2 
 15.8 15.0  95  15.3  97  15.1 
 5.00 5.12  102  4.99  100  5.05 
 1.58 1.81 114  1.50  95 1.65 
 0.500 0.572  114  0.456 

91

0.511 

 0.158

0.190 

120 

0.125 

79 

0.154 

Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline. Details are given in the executive summary.
Conclusions:
In this study (OECD 201; GLP), tri-n-butylamine was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean test item concentrations):

The EC50 -values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 10.1 (9.38 -10.9) mg/L and 5.13 (4.14 -7.26) mg/L, respectively.
The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.65 mg/L and 0.154 mg/L, respectively.
The EC10 value with 95% confidence interval for inhibition of growth rate (ErC10) after 72 hours was 4.64 (3.97 – 5.53) mg/L.
Executive summary:

The toxicity of tri-n-butylamine to the unicellular freshwater green algae Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018 -06 -11 to 2018 -06 -15 at the test facility, with the definitive exposure phase from 2018 -06 -12 to 2018 -06 -15. The aim of the study was the determination of NOEC, LOEC, EC10 -, EC20 -, and EC50 -values of growth rate and yield over a period of 72 hours.

This GLP-compliant study was conducted under static conditions with an inital cell density of 6442 cells/mL. A stock solution of 50.0 mg/L was prepared with dilution water. Six concentrations in a geometrical series with a dilution factor of 10 were tested (nominal): 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L, corresponding to the geometric mean measured test item concentrations 0.154 - 0.511 - 1.65 - 5.05 - 15.1 - 43.2 mg/L.

The concentrations of the test item tri-n-butylamine were analytically verified by LC-MS/MS at the start and at the end of the exposure of all test concentrations and the control. The measured concentrations of the test item were between 88 and 120% of the nominal value at the start of the exposure (0 hours) and between 79 to 100% of the nominal value at the end of the exposure (72 hours). Therefore, all effect values given are based on the geometric mean measured test item concentrations of tri-n-butylamine.

The EC50 -values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 10.1 (9.38 -10.9) mg/L and 5.13 (4.14 -7.26) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.65 mg/L and 0.154 mg/L, respectively.

The EC10 value with 95% confidence interval for inhibition of growth rate (ErC10) after 72 hours was 4.64 (3.97 – 5.53) mg/L.

The study meets the validity criteria of the guideline:

  • Increase of the cell growth in the control cultures: 285-fold (specific growth rate 1.88 day-1) compared to a requirement of at least 16 (specific growth rate 0.92 day-1)
  • Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures: 18.8 % compared to a requirement of ≤ 35%
  • Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures: 1.63% compared to a requirement of ≤ 7%
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test procedure in accordance with national standard methods (DIN 38412, part 9); detailed study report not available; however, the data were peer-reviewed by BUA and have been used in a legally binding classification (KBwS, WGK 2); hence, effect values are considered as reliable
Qualifier:
according to
Guideline:
other: German Industrial Standard DIN 38412, part 9 (draft)
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
pH:
pH 10.5; test medium not neutralised
Details on test conditions:
- algae test apparatus with quasi-continuous determination of optical density      
- photometric determination of turbidity at pre-determined times     
- test solutions tested non-neutralized and neutralized
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.477 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions not neutralised; evaluated presumably on biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.556 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions not neutralised; evaluated presumably on biomass
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
1.177 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions not neutralised; evaluated presumably on biomass
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.91 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions not neutralised; evaluated presumably on biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.378 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions neutralised; evaluated presumably on biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.215 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions neutralised; evaluated presumably on biomass
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
1.199 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions neutralised; evaluated presumably on biomass
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
5.578 mg/L
Nominal / measured:
not specified
Basis for effect:
biomass
Remarks on result:
other: test solutions neutralised; evaluated presumably on biomass
Validity criteria fulfilled:
not specified
Conclusions:
Desmodesmus subspicatus, endpoint biomass:
72-h EC50 = 8.2 mg/L (test solutions neutralised)
72-h EC10 = 1.4 mg/L (test solutions neutralised)

Desmodesmus subspicatus, endpoint biomass:
72-h EC50 = 3.6 mg/L (test solutions)
72-h EC10 = 1.5 mg/L (test solutions)
Executive summary:

The growth inhibiting effect of tributylamine was investigated on the green alga Desmodesmus subspicatus in a 96 -h study according to German Industrial Standards DIN 38412, part 9 (draft). In part A) of the experiment, the test solutions were not neutralised. The endpoint was growth (biomass). The EC50 was determined to be 3.6 mg/L after 72 h and 2.9 mg/L after 96 h. The EC10 was determined to be 1.5 mg/L after 72 h, 1.2 mg/L after 96 h.

After neutralization of test solutions in part B) of the experiment, the following effect values were observed: EC50 (72 h) 8.2 mg/L and EC10 (72 h) 1.38 mg/L; EC50 (96 h) 5.8 mg/L and EC10 (96 h) 1.2 mg/L.

Description of key information

A valid OECD 201 (GLP; RL 1) study with Pseudokirchneriella subcapitata over 72 hours was performed (Herrmann, 2018) with analytical verification of test item concentrations.

The following ECx -values with 95% confidence intervals were determined (based on geometric mean measured test item concentrations):

  • ErC50: 10.1 (9.38 -10.9) mg/L
  • EyC50: 5.13 (4.14 -7.26) mg/L
  • ErC10: 4.64 (3.97 – 5.53) mg/L

The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.65 mg/L and 0.154 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
10.1 mg/L
EC10 or NOEC for freshwater algae:
4.64 mg/L

Additional information

In the valid key study (Herrmann, 2018; RL 1) the toxicity of tri-n-butylamine to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined compliant with GLP according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. over 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A stock solution of 50.0 mg/L was prepared with dilution water. Six concentrations in a geometrical series with a dilution factor of √10 were tested (nominal): 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L, corresponding to the geometric mean measured test item concentrations 0.154 - 0.511 - 1.65 - 5.05 - 15.1 - 43.2 mg/L.

The concentrations of the test item tri-n-butylamine were analytically verified by LC-MS/MS at the start and at the end of the exposure of all test concentrations and the control. The measured concentrations of the test item were between 88 and 120% of the nominal value at the start of the exposure (0 hours) and between 79 to 100% of the nominal value at the end of the exposure (72 hours). Therefore, all effect values given are based on the geometric mean measured test item concentrations of tri-n-butylamine.

The EC50 -values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 10.1 (9.38 -10.9) mg/L and 5.13 (4.14 -7.26) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 1.65 mg/L and 0.154 mg/L, respectively.

The EC10 value with 95% confidence interval for inhibition of growth rate (ErC10) after 72 hours was 4.64 (3.97 – 5.53) mg/L.

All validity criteria of the guideline were met.

Supporting data are available:

The assessment of the algal toxicity according to Nusch (1987) is based on effect data taken from the “Commission for the Evaluation of Substances Hazardous to Waters” (KBwS) data sheet no. 594, which is part of the German Federal Water Act (Article 19g). Based on the collected data a substance is classified according to its water-hazardous properties (WGK 1 to 3). Tributylamine was classified as WGK 2, which indicates a medium hazard to waters. The WGK 2 classification was triggered by the algal toxicity. As the list with the classified substances was published in the Bundesanzeiger, the classification is legally binding for the industry.

The data sheet lists only one source for effect data on algae (test with neutralised and non-neutralised test solutions). These data were generated within the research project “Assessment of Water-Hazardous Substances” which served the scientific support and administration of a “Commission” in their work of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety. The data were peer-reviewed by BUA and published in an official report. Hence, the result can be regarded as reliable (RL 2).

The algal toxicity tests were performed according to DIN 38412, part 9 (draft). In case of tributylamine, the tests were performed with neutralised and non-neutralised test medium in parallel. The test species was Scenedesmus subspicatus (new name: Desmodesmus subspicatus). The EC10 was determined being the relevant factor for the WGK classification. The effect data were presumably evaluated based on biomass, although there is no clear indication on the evaluation base.

The effect values show a higher toxicity using non-neutralised test medium with regard to the EC50; therefore, part of the observed toxicity is caused by the pH change induced by the substance. For the risk assessment the effect data for the neutralised test medium should be used since the quantities of TBA that would be found in natural waters are not likely to affect the pH to a relevant extent. It is likely that the EbC50 value from the test without neutralisation overestimates the potential toxicity of these matrices.

Detailed data on the study itself, e.g. biomass growth rate as well as growth curves are not available. Nevertheless the test results were performed according to national standards and peer-reviewed by the KBwS during the performance of the research project. Therefore, the data can be regarded as reliable with restrictions (RL 2).

This test was performed without analytical monitoring of test item concentrations. To account for this, TOC measurements in screening tests (without organisms) according to OECD 201 (>80% recovery after 96 h) and OECD 202 (>80% recovery after 48 h) were performed (BASF, 2014). The results confirm stability of n-butylamine over the duration of the test. The following results were determined according to Nusch (1987):

Desmodesmus subspicatus, 72 hours ECx (biomass), based on nominal concentrations:

EbC50 neutralized/not neutralized: 8.2/3.6 (Nusch (1987), cited in Amann et al. (1987/1988)

EbC10 neutralized/not neutralized: 1.4/1.5 (Nusch (1987), cited in Amann et al. (1987/1988)

Overall conclusions on algal toxicity data:

The EC50 (72 h; nominal) based on yield of 8.2 mg/L (neutralized test item solutions; Nusch, 1987) is fairly close to the corresponding effect concentration determined in the valid key study of 5.13 mg/L based on geometric mean measured concentrations (Herrmann, 2018). As such, data from the supporting study corroborate data determined in the valid key study.