4-dimethylaminobenzaldehyde

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:

July 16, 2015 - January 8, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-dimethylaminobenzaldehyde

EC Number:

202-819-0

EC Name:

4-dimethylaminobenzaldehyde

Cas Number:

100-10-7

Molecular formula:

C9H11NO

IUPAC Name:

4-(dimethylamino)benzaldehyde

Test material form:

solid

Specific details on test material used for the study:

Designation: Art. 803057
Synonym: 4-(Dimethylamino)benzaldehyde
CAS-No.: 100-10-7
Batch: S6886257
Assay (GC, area%): 99.6% (a/a)
Appearance: Grey-blue, crystalline powder
Expiry date: May 31, 2019
Storage: Tightly closed, dark at room temperature (15 to 25°C)

PREPARATION OF THE TEST MATERIAL
The test item was prepared freshly before the exposure. For this purpose, the calibrated flask with test medium (100 mg/L) was stirred for 24 hours.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentrations:
5.0, 10.6, 22.4, 47.3 and 100.0 mg/L

Sampling method:
Samples of the test medium and the control medium were taken directly after preparation and after 24, 48 and 72 hours and were subjected to chemical analysis. Before analysis, the algae were removed from the medium by centrifugation at a low g-force, sufficient to settle the algae. Additionally, analysis of all five test item preparations not inoculated with algae was performed daily.

Sample storage conditions before analysis:
Samples were directly analyzed without storing

Test solutions

Vehicle:

yes

Details on test solutions:

Vehicle:

Nutrient (mg/L)
NH4Cl 15.0
MgCl2 * 6H2O 12.0
CaCl2 * 2H2O 18.0
MgSO4 * 7H2O 15.0
KH2PO4 1.60

FeCl3 * 6H2O 0.0640
Na2EDTA * 2H2O 0.100

H3BO3 0.185
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2 * 6H2O 0.00150
CuCl2 * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700

NaHCO3 50.0

- pH: 7.70
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
The test item was prepared freshly before the exposure. For this purpose, the calibrated flask with test medium (100 mg/L) was stirred for 24 hours.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Desmodesmus subspicatus
- Source (laboratory, culture collection): The test system used was Desmodesmus subspicatus, strain 86.81 SAG (Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany). This strain was further cultivated in the laboratories of Non-Clinical Safety.
- Method of cultivation: Preparations of 100 mL each were continuously shaken in 300 mL Erlenmeyer flasks at about 120 rpm by an orbital shaker (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany). The flasks were covered with air permeable steristoppers (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The flasks were continuously illuminated for the exposure period. The anticipated light intensity was between 4440 and 8880 Lux and within ± 15% from the average. The illumination was achieved by fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, Munich, Germany) installed above the orbital shaker with the flasks.

ACCLIMATION
- Acclimation period: 72h
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

-

Test conditions

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

22.7 - 23.4°C

pH:

7.48 - 9.36

Nominal and measured concentrations:

Nominal concentrations: 5.0, 10.6, 22.4, 47.3 and 100.0 mg/L
Measured concentrations: >80% of nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glas flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 cells per ml
- Control end cells density: 1 744 672 cells per ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The light intensity measured immediately before the start of the study was 8175 Lux ± 4.6% and 8216 Lux ± 5.3% at the end of the study. The light intensity for the samples used for analytical determination after 24 and 48 hours was 8496 Lux ± 2.2% before the start of the study and
8458 Lux ± 1.8% after 48 hours. Thus, the light intensity was in the anticipated range of 4440 – 8880 Lux within ± 15% from the average.


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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.115
- Test concentrations: 5.0, 10.6, 22.4, 47.3 and 100 mg/L

Reference substance (positive control):

no

Remarks:

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control (Gado, 2015).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

42.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC10 based on nominal concentrations

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

50.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC20 based on nominal concentrations

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

72.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC50 based on nominal concentrations

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

19.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: EC10 based on nominal concentrations

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

25 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: EC20 based on nominal concentrations

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: EC50 based on nominal concentrations

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control (Gado, 2015).

The test item Art. 104864 (potassium dichromate) showed 72h EC values which are within the recommended range (Biomass integral EC50 0.20 – 0.75 mg/L and Growth rate EC50 of 0.60 – 1.03 mg/L).

Any other information on results incl. tables

Objective

The objective of this study was to evaluate the influence of the test item Art. 803057 (4-(Dimethylamino)benzaldehyde) on the growth and growth rate of the green algae species Desmodesmus subspicatus.

Study Design

The study design included one control group with six replicates and five test item groups with three replicates, each containing 100 mL reconstituted water or test medium and 10000 cells/mL at the start of the experimental phase.

The study was performed under static conditions with nominal concentrations of 5.0, 10.6, 22.4, 47.3 and 100.0 mg/L in reconstituted water. The study was performed in open vessels.

The growth of the algae was determined after 24, 48, and 72 hours exposure in the test item and control groups.

The concentrations of the test item Art. 803057 (4-(Dimethylamino)benzaldehyde) were analytically determined by HPLC-UV. Due to the results of a stability determination before start of study, analytical controls of the test item preparations were performed at the start of the study and then daily.

Results

Nominal and corresponding analytical concentrations of samples inoculated with algae are given in the following table:

	Analytical results
	0h		24h		48h		72h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%	[mg/L]	%
0.0 mg/L	<LOD	-	<LOD	-	<LOD	-	<LOD	-
5.0 mg/L	4.974	99.5	4.192	83.8	0.276	5.5	<LOD	-
10.6 mg/L	10.608	100.1	9.971	94.1	7.957	75.1	2.286	21.6
22.4 mg/L	22.122	98.8	21.645	96.6	20.621	92.1	17.907	79.9
47.3 mg/L	46.806	99.0	46.003	97.3	45.221	95.6	43.262	91.5
100.0 mg/L	100.146	100.1	98.892	98.9	98.458	98.5	97.950	98.0

Nominal and corresponding concentrations of analytical control samples without algae are given in the following table:

	Analytical results
	0h		24h		48h		72h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%	[mg/L]	%
0.0 mg/L	<LOD	-	<LOD	-	<LOD	-	<LOD	-
5.0 mg/L	4.974	99.5	4.973	99.5	4.933	98.7	4.866	97.3
10.6 mg/L	10.608	100.1	10.644	100.4	10.584	99.8	10.523	99.3
22.4 mg/L	22.122	98.8	22.219	99.2	22.194	99.1	22.171	99.0
47.3 mg/L	46.806	99.0	46.843	99.0	46.959	99.3	46.856	99.1
100.0 mg/L	100.146	100.1	100.072	100.1	100.104	100.1	100.187	100.2

The recoveries of the Art. 803057 concentrations in all test media without algae ranged from 97.3% to 100.4% from the nominal concentrations over the time period of 72h and, thus, the test item was stable in the test medium during the study. In contrast, the concentrations of the test item in test media with algae decreased significantly after 24, 48 and 72 hours particularly at lower concentrations. Based on the analytical results it can be assumed that a fraction of the test item in the test media adsorbs onto the surface of the algae during the study and, therefore, is still bioavailable for the test system. Moreover, there are indications from the HPLC analysis that the test item is metabolized by the algae to a certain extent confirming the bioavailability of the test item for the algae.

Therefore, it is reasonable to conclude, that the test item is chemically stable in the test medium and that the algae are exposed to the test item at concentrations of ± 20% of the nominal concentrations during the experimental phase of the study. Therefore, the EC values were calculated based on the nominal concentrations.

For the test item, Art. 803057 (4-(Dimethylamino)benzaldehyde), the following EC values (growth rate and yield) for Desmodesmus subspicatus were determined:

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	42.2 (39.3 - 44.8)	19.3 (14.7 - 23.0)
EC20 [mg/L] 95% confidence interval	50.8 (48.2 - 53.3)	25.0 (20.6 - 28.6)
EC50 [mg/L] 95% confidence interval	72.7 (70.2 - 75.2)	41.3 (37.2 - 45.8)

Conclusion

Art. 803057 (4-(Dimethylamino)benzaldehyde), dissolved in reconstituted water, was tested in an open semi-static system. Under the conditions of the present study the test item revealed an EC 50(0-72h) for growth rate of 72.7 mg/L and an EC50 (0-72h) for yield of 41.3 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Art. 803057 (4-(Dimethylamino)benzaldehyde), dissolved in reconstituted water, was tested in an open semi-static system. Under the conditions of the present study the test item revealed an EC50 (0-72h) for growth rate of 72.7 mg/L and an EC50 (0-72h) for yield of 41.3 mg/L.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201. Art. 803057 (4-(Dimethylamino)benzaldehyde), dissolved in reconstituted water, was tested in an open semi-static system. Under the conditions of the present study the test item revealed an EC50 (0-72h) for growth rate of 72.7 mg/L and an EC50 (0-72h) for yield of 41.3 mg/L.