Dimethylamine-borane (1:1)

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

Study period:

2017-10-24 to 2018-01-29

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)

Version / remarks:

adopted March 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at 4 ± 4 °C, in the dark

Analytical monitoring:

yes

Details on sampling:

Substance specific analysis was performed at the test site of WeylChem InnoTec GmbH
- Concentrations: 0, 0.25, 0.74, 2.2, 6.7, 20 mg/L (nominal loading rate)
- Sampling method: Triplicate samples from the freshly prepared test medium of all test concentrations and the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, triplicate samples from the test medium of all test concentrations and the control were collected at the end of the test (after 72 hours).
- Sample storage conditions before analysis: All samples were stored in a in a refrigerator (4 ± 4 °C) immediately after sampling, protected from light until shipment.
Two replicates of the samples of the only test concentration and the control were shipped from ibacon to the test site of WeylChem InnoTec. Samples were shipped at ambient temperature (+5 to 30 °C) to WeylChem InnoTec.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 20 mg test item/L was prepared by dissolving 20.0 mg test item into 1000 mL test water by intense stirring for a short period. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the algae (= start of the test).
- Controls: yes, reconstituted water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): not reported
- Method of cultivation: under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: The test was started (0 hours) by inoculation of a biomass of approx. 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Culturing media and conditions (same as test or not): 3 days before test the same culturing conditions as in the test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.0 - 22.5 °C

pH:

at the beginning of the test: 8.1 - 8.2
at the end of the test: 8.0 - 9.5

Dissolved oxygen:

not applicable

Salinity:

not applicable

Conductivity:

no data

Nominal and measured concentrations:

Nominal loading rate in the test vessel [mg/L]: 0, 0.25, 0.74, 2.2, 6.7, 20

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium
- Type (delete if not applicable): The flasks were covered with glass dishes and incubated in a water bath.
- Material, size, headspace, fill volume: 50 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no, static test
- Initial cells density: At test start approx. 5000 algal cells/mL were incubated per test vessel
- Control end cells density: 926,990 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, the algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

TEST MEDIUM / WATER PARAMETERS
Test Water
Reconstituted Water (OECD Medium): Analytical grade salts were added at the following nominal concentrations in deionised water:
Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 X 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 X 7 H2O 15.0 mg/L
MgSO42 X 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace elements:
Na2EDTA X 2 H2O 100.0 µg/L
FeCl3 X 6 H2O 64.0 µg/L
MnCl2 X 4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4 X 2 H2o 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 X 6 H2O 1.5 µg/L
CuCl2 X 2 H2O 0.01 µg/L
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
- Intervals of water quality measurement: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The pH was measured in all test item concentrations and the control at the start and the end of the test. The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 5103 lux (4890 to 5410 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density, every 24 h; The inhibition of algal growth was determined from yield and growth rate; calculation according to the OECD guideline 201
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.
- Chlorophyll measurement: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: Yes. Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
The pre-experiments were not performed in compliance with the GLP-Regulations and are be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study number of the present study.
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 6.29 - 8.95 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 3.35 - 7.35 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI: 2.33 - 7.12 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 10.6 - 12.6 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 7.41 - 8.95 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 6.05 - 7.56 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 2.2 mg/L and the algal cells in the control. At the concentration of 6.7 mg/L and above the algal cells were smaller than in the control and showed deformations.
- 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? Yes
- EC50: (Growth Rate) 1.02 mg/L (95%-CI: 0.984 - 1.05 mg/L)

Reported statistics and error estimates:

The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Analytical results:

The quantification of the analyte boron of the test item dimethylamine-borane in the test samples was performed using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). At the start of the test recoveries of the nominal test concentration varied between 99 and 113 % (test concentrations of nominal 2.2, 6.7 and 20 mg test item/L considered). After 72 hours test duration, the recoveries of the nominal values varied between 99 and 116 % (test concentrations of nominal 2.2, 6.7 and 20 mg test item/L considered). The lowest test concentrations of 0.74 and 0.25 mg test item/L lie below the LOQ of the analytical method and could not be were analytically quantified. However, the measured concentrations of boron significantly exceeded the theoretically possible concentrations of 0.13 and 0.046 mg B/L at 0.74 and 0.25 mg test item/L, respectively. These increased boron concentrations are most probably attributable to relatively high leaching of boron from the sample glass containers in relation to the low boron concentrations originating from the test item itself.

Conclusion:

The influence of dimethylamine-borane on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 7.50 mg test item/L and the 72-hour ErC50 value was calculated to be 11.6 mg test item/L. The 72-hour NOEyC was determined to be 2.2 mg test item/L and the associated 72-hour LOEyC was 6.7 mg test item/L. The 72-hour NOErC was determined to be 2.2 mg test item/L and the associated 72-hour LOErC was 6.7 mg test item/L.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. Stability and maintenance of three highest exposure concentrations of nominal 2.2, 6.7 and 20 mg test item/L could be demonstrated throughout the 72-h exposure duration, as the quantifiable concentrations were within ± 20 % of the nominal concentrations. It is assumed that the same applies also to the two lowest test concentrations. Therefore, all reported results refer to nominal values. Since the quantifiable concentrations of the test item were within ± 20 % of the nominal concentrations during the test, assuming that the lowest two test concentrations below the LOQ also fulfil this criterion.

Validity Criteria:

Cell Density - Increase in the Control Cultures: 185.4-fold increase within 72 hours and thus, the validity criterion was met.

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 6.4 % and thus, the validity criterion was met.

Coefficient of Variation of Average Growth between Control Replicates: 2.3 % and thus, the validity criterion was met.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 of the test item in Pseudokirchneriella subcapitata is 11.6 mg/L (95 % C.I. 10.6 - 12.6 mg/L; nominal values).

Executive summary:

In a 72 hour acute toxicity study, cultures of Pseudokirchneriella subcapitata, Strain No. 61.81 SAG were exposed to dimethylamine-borane at nominal concentrations of 0, 20, 6.7, 2.2, 0.74 and 0.25 mg test item/L under static conditions in accordance with the OECD guideline 201. The NOE_rC and E_rC₅₀ values based on growth rate were 2.2 and 11.6 mg/L, respectively.

The following abnormalities were noted: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 2.2 mg test item/L and the algal cells in the control. At the concentration of 6.7 mg/L and above the algal cells were smaller than in the control and showed deformations.

This toxicity study is classified as acceptable and satisfies the guideline requirements for algae growth inhibition study according to OECD guideline 201.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type (Flowthrough, Static, Static Renewal): Static

 

Effect levels:

72 hr EC10: 6.75 mg a.i./L                95 % C.I.: 6.05 to 7.56 mg a.i./L

72 hr EC50: 11.6 mg a.i./L                95 % C.I.: 10.6 to 12.6 mg a.i./L

72 hr NOEC: 2.2 mg a.i./L                  

 

Endpoint(s) Effected: growth rate

Description of key information

In a 72-hour algae growth inhibition test cultures of Pseudokirchneriella subcapitata were exposed to dimethylamine-borane at nominal concentrations of 0.0 (control), 20, 6.7, 2.2, 0.74 and 0.25 mg test item/L under static conditions in accordance with the OECD 201 (2006) and GLP.

The following effect levels - based on the growth rate - are calculated:

- 72 h ErC50: 11.6 mg/L (95 % C.I. 10.6 – 12.6 mg/L);

- 72 h NOErC: 2.2 mg/L and

- 72 h LOErC: 6.7 mg/L.  

Key value for chemical safety assessment

EC50 for freshwater algae:

11.6 mg/L

EC10 or NOEC for freshwater algae:

2.2 mg/L