N,N-Bis-(4-biphenyl)amine

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:

Sep 18 - Nov 04, 2019

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)

Analytical monitoring:

yes

Details on sampling:

The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director.
Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from 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, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.

Vehicle:

no

Details on test solutions:

Biological treatment samples and control samples:
The samples were allowed to thaw to room temperature. They were then shaken well and treated with ultrasound for 1 minute to obtain homogenous samples. The samples were already diluted with acetonitrile by factor 2 directly after sampling. The samples were centrifuged (13,000 rpm, 3 minutes) before analysis to remove the algae.
Fortified samples and analytical blank control samples:
The samples were shaken well and were centrifuged (13,000 rpm, 3 minutes) before analysis.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: 61.81 SAG
- Source (laboratory, culture collection): "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L

Test temperature:

21.7 to 22.6 °C

pH:

7.9 to 8.0 at test start
9.3 to 9.5 at test end

The pH was adjusted with 1 M NaOH to pH 8.1

Nominal and measured concentrations:

nominal: 100 mg/L
analytical: below LOQ of 0.005 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

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): none
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: -
- Any stimulation of growth found in any treatment:
- 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):

Parameter (0-72 h)
EC50 (yield) = 0.445 mg/L
EC50 (growth rate) = 0.941 mg/L
EC50 (biomass) = 0.494 mg/L

Influence of the test material on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Yield [mg test item/L]	Growth rate [mg/L]
72-hour EC50	> 100	> 100
95 % conf. interval	n.d.	n.d.
72-hour EC20	> 100	> 100
95 % conf. interval	n.d.	n.d.
72-hour EC10	> 100	> 100
95 % conf. interval	n.d.	n.d.
72-hour NOEC	≥ 100	≥ 100
72-hour LOEC	> 100	> 100

CI: Confidence interval n.d.: not determinable NOEC and LOEC were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hourEC10 and the 72-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L, which is the concentration representing the limit of solubility.
The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. No test item concentrations above the limit of quantification of the analytical method were found. All reported results refer to the nominal concentrations.

Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata.
For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.
The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of the recommended test species.
The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement. Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested, corresponding to nominal concentrations of 100, 50, 25, 12.5 and 6.25 mg test item/L. All concentrations were below the limit of quantification of the analytical method (LOQ of 0.005 mg test item/L). No concentrations above the solubility limit of the test item in the used test water were tested. Additionally, a control was tested in parallel.
The exposure was analytically verified using an HPLC method with UV detection.

Biological Results

The 72 -h EC50 and 72 -h EC10 values were determined to be > 100 mg/L based on growth rate.

Values refer to nominal test concentrations of a filtrate of 100 mg test item/L, which were measured below the limit of quantification of the analytical method (LOQ of 0.005 mg test item/L)

Analytical Results

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. In the test media of the only test concentration (filtrate of test item stock solution of nominal 100 mg test item/L) the measured values are below the limit of quantification of the analytical method (LOQ = 0.005 mg test item/L).

Conclusion

The influence of the test irem on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC and the 72-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of the test item up to the filtrate of nominal 100 mg test item/L, which is the concentration representing the limit of solubility.
The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. No test item concentrations above the limit of quantification of the analytical method were found. All reported results refer to the nominal concentrations.

Description of key information

OECD 201:

No adverse effects of the test item to algae were observed up to the limit of water solubility.

EC50 (growth rate, 72h) > 100 mg/L (nominal)

EC10 (growth rate, 72h) > 100 mg/L (nominal)