Benzaldehyde

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:

2018-12-03 to 2018-12-10

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:

OECD Guideline No. 201 for Testing of Chemicals (2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 266/2016/Method C.3

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: Benzaldehyde
CAS No./EC No.: 100-52-7/202-860-4
Batch number: #1803-1
Purity (certified): > 99.5 % wt
Appearance: Clear, colorless to light yellow, liquid
Water solubility: 6.95 g/L at 25 °C
Relative density: 1.041 – 1.046 at 25 °C
Vapor pressure: 169 Pa at 25 °C
Expiry date: 2020-01-15

Analytical monitoring:

yes

Details on sampling:

Chemical Analysis of Test Item Concentrations

Determination of the test item: The samples were analysed under GLP with a GC-MS method, which has been implemented under non-GLP and documented finally in the GLP raw data.

Sampling schedule: All concentration levels and the control were analytically verified via GC-MS after 0 hours (start of exposure) and 72 hours (end of the exposure) with algae.

Sampling and pre-treatment: Separate replicates for the test item analysis after 0 hours and 72 hours were prepared in the original test vessels with algae at the start of the exposure and incubated under test conditions until analysis. The samples were stabilized immediately after sampling by extraction. The samples were preferably measured within one day after sampling.

Biological Parameters

Chlorophyll a-fluorescence: 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 100 mg/L test item solution.

Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Physical Chemical Properties

The pH-values at the start of exposure were measured in one additional replicate of each concentration level and the control. At the end of exposure, the pH-values were measured from pooled samples of the test item concentrations and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

Equipment
pH-Meter, pH 191 (WTW)
Hygro-thermograph (KLIMATHERM)
Digital Lux meter (TESTO)
Fluorometer: Microplate Reader Chameleon V (HIDEX) with Software Micro Win 4.41 (MIKROTEC LABORSYSTEME GMBH) for determination of the cell density
Shaker, SM 25 A (BÜHLER)
Light microscope (ZEISS Lab 16)
Fluorescent tubes, OSRAM L 36 W/865, cool daylight (test), OSRAM L 30 W/840, cool white energy saver (culture)
Piston Stroke Pipettes (THERMO SCIENTIFIC, BRANDT)
Analytical balance, LA230-S (SARTORIUS)
Balance, PLS 1200-3A (KERN)

Vehicle:

no

Details on test solutions:

Test item: Benzaldehyde

Stock solution, media preparation: A stock solution with a nominal test item concentration of 50.0 mg/L was freshly prepared with dilution water prior to the start of exposure. The stock solution was agitated until the solution was visually clear.

Test concentrations (nominal): Seven concentration levels were tested in a geometrical series with (nominal) dilution factor of √10: 0.0500 - 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0140 – 0.0235 – 0.0867 – 0.104 – 0.174 – 7.28 – 42.4 mg/L. The concentration levels are based on the results of a preliminary range finding test.

Control: Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Reference Item: Potassium dichromate was tested as a reference item.

Replicates: Three replicates per concentration level and six for the control were tested. Separate replicates for each measuring time were prepared.

Test containers: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals

Test volume: 59 mL

Test organisms (species):

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

Details on test organisms:

Test organism: Pseudokirchneriella subcapitata HINDÁK CCAP 278/4

Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)

Reason for the selection of the test organism: Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.

Origin: Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.

Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).

Preculture: A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.

Initial cell density Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6442 cells/mL

Test type:

static

Test temperature:

21 – 24 °C

Details on test conditions:

Test method: Static procedure

Performance of the test: A static test was carried out. With regard to the volatility of the test item sealed glass flasks without headspace were used to reduce loss of test item.

Duration of the test: 72 hours

Replicates: Three replicates per concentration level and six for the control were tested. Separate replicates for each measuring time were prepared.

Test containers: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals

Test volume: 59 mL

Preculture: A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.

Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6442 cells/mL

Application: Application was carried out by adding appropriate volumes of algae inoculum to the test solutions.

Incubation: The flasks were positioned randomly and repositioned daily.

Temperature (target): Nominal range: 21 – 24 °C, controlled at ± 2 °C

Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

Light intensity (target): Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1

Light regime: 24 hours/day light

Light homogeneity (target): Within ± 15% over incubation area

Reference substance (positive control):

yes

Remarks:

Potassisum dichromate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

growth rate

Effect conc.:

33.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Remarks:

growth rate

Effect conc.:

0.039 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Yield

Effect conc.:

8.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Yield

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The toxicity of Benzaldehyde (Batch no.: #1803-1) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018-12-03 to 2018-12-10 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL. A stock solution with a nominal concentration of 50.0 mg/L was prepared once prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was agitated until it was visually clear. Seven concentration levels were tested in a geometrical series with a spacing factor of √10: 0.0500 - 0.158 - 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0140 – 0.0235 – 0.0867 – 0.104 – 0.174 – 7.28 – 42.4 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

At the start of the exposure the measured concentrations were in the range of 84 to 98% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 80% of the nominal test item concentrations.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, 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 2018-09-25 to 2018-09-28 (with headspace) and 2018-07-10 to 2018-07-13 (without headspace), respectively. The reference item toxicity is in the valid range following test facility SOPS.

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
42.4	1	6442	12487	18805	33087
	2	6442	11385	18524	38019
	3	6442	11596	19726	33561
	Mean	6442	11823	19018	34889
7.28	1	6442	14438	60431	227201
	2	6442	17819	55625	207966
	3	6442	17636	51489	261122
	Mean	6442	16631	55848	232096
0.174	1	6442	18626	55039	193548
	2	6442	16710	55481	209972
	3	6442	14813	57532	182367
	Mean	6442	16716	56017	195296
0.104	1	6442	17201	54404	229326
	2	6442	18346	58704	167571
	3	6442	17479	50114	224470
	Mean	6442	17675	54407	207122
0.0867	1	6442	23350	93598	330791
	2	6442	22117	82178	354300
	3	6442	21474	107321	339479
	Mean	6442	22314	94366	341523
0.0235	1	6442	29000	119738	376940
	2	6442	26846	122410	363311
	3	6442	28010	127662	437786
	Mean	6442	27952	123270	392679
0.0140	1	6442	29867	117713	403553
	2	6442	25108	114570	393325
	3	6442	25145	117226	412291
	Mean	6442	26707	116503	403056
Control	1	6442	30371	124892	454916
	2	6442	29418	136033	403900
	3	6442	29341	133611	402584
	4	6442	29517	134733	410578
	5	6442	28137	127280	441907
	6	6442	30746	125627	398513
	Mean	6442	29588	130363	418733

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-). 

Geometric mean measured test item concentration	Replicate	Growth Rate		Inhibition of Growth Rate	Yield		Inhibition of Yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
42.4	1		0.55	61		26645	94
	2		0.59	57		31577	92
	3		0.55	60		27119	93
	Mean	(+)	0.56	60	(+)	28447	93
7.28	1		1.19	15		220759	46
	2		1.16	17		201524	51
	3		1.23	11		254680	38
	Mean	(+)	1.19	14	(+)	225654	45
0.174	1		1.13	18		187106	55
	2		1.16	17		203530	51
	3		1.11	20		175925	57
	Mean	(+)	1.14	18	(+)	188854	54
0.104	1		1.19	14		222884	46
	2		1.09	22		161129	61
	3		1.18	15		218028	47
	Mean	(+)	1.15	17	(+)	200680	51
0.0867	1		1.31	6		324349	21
	2		1.34	4		347858	16
	3		1.32	5		333037	19
	Mean	(+)	1.32	5	(+)	335081	19
0.0235	1		1.36	2		370498	10
	2		1.34	3		356869	13
	3		1.41	-1		431344	-5
	Mean	(-)	1.37	2	(-)	386237	6
0.0140	1		1.38	1		397111	4
	2		1.37	1		386883	6
	3		1.39	0		405849	2
	Mean	(-)	1.38	1	(-)	396614	4
Control	1		1.42			448474
	2		1.38			397458
	3		1.38			396142
	4		1.38			404136
	5		1.41			435465
	6		1.37			392071
	Mean		1.39			412291

Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.55	1.41	1.29	1.42	0.129	9.10	14.5
	2	1.52	1.53	1.09	1.38	0.252	18.3
	3	1.52	1.52	1.10	1.38	0.239	17.3
	4	1.52	1.52	1.11	1.39	0.234	16.9
	5	1.47	1.51	1.25	1.41	0.144	10.2
	6	1.56	1.41	1.15	1.38	0.206	15.0
				Mean	1.39
				SD ±	0.02
				CV [%]	1.33

SD = Standard deviation  CV = Coefficient of variation

Physicochemical Data

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits in the definitive study. The test media were clear throughout the exposure phase.

 

Environmental Conditions

Geometric mean measured test item concentration		pH-values
[mg/L]		Start; 0 hours	End; 72 hours
42.4		8.24	8.31
7.28		8.25	8.05
0.174		8.24	8.56
0.104		8.21	8.80
0.0867		8.18	9.16
0.0235		8.15	9.16
0.0140		8.16	9.14
Control		8.19	9.10
Room temperature [°C]:	min.: 22.5	max.: 23.5	mean value: 23.0
Light intensity [lux]	n = 23	mean value: 5744 range of the measured values: 5025 to 6261 equalling -12.5 to 9.00%	CV [%]: 6.84%

CV = Coefficient of variation                                        n = number of measuring points

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of Benzaldehyde and the control were determined in fresh media (0 hours) and at the end of the exposure interval (72 hours) via GC-MS.

At the start of the exposure the measured concentrations were in the range of 84 to 98% of the nominal values. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 80% of the nominal test item concentrations. The geometric mean measured concentrations are 42.4 – 7.28 – 0.174 – 0.104 – 0.0867 – 0.0235 – 0.0140 mg/L.

Measured Concentration of Benzaldehyde in the Definitive Test with Algae

Sampling date	Fresh Media, 0 hours		Old Media, 72 hours
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Geometric mean measured concentration^1) [mg/L]
	Benzaldehyde
50.0	44.8	90	40.1	80	42.4
15.8	14.8	94	3.58	23	7.28
5.00	4.75	95	0.00640*	0	0.174
1.58	1.41	89	0.00774*	0	0.104
0.50	0.419	84	0.0179	4	0.0867
0.158	0.138	87	< LOQ	-	0.0235
0.050	0.0490	98	< LOQ	-	0.0140
Control	< LOQ	-	< LOQ	-	< LOQ

Meas. conc.= measured concentration of the test item, dilution factors taken into account

% = percent of the nominal measured concentration of the test item

LOQ = limit of quantification of the analytical method (0.008 mg/L of the test item)

1) = Values < LOQ taken into account with ½ LOQ (0.004 mg/L of the test item)

* = < LOQ but > 70% LOQ

NOEC, LOEC, EC_x-values ofBenzaldehyde(0 - 72 hours)

                  (based on the geometric mean measured test item concentrations, with 95% confidence intervals)

	Growth Rate Inhibition [mg/L]
NOEC	0.0235
LOEC	0.0867
ErC10	0.0389 (0.0258 – 7.62)
ErC20	9.70 (5.79 – 13.3)
ErC50	33.1 (28.2 – 39.5)
	Yield Inhibition  [mg/L]
NOEC	0.0235
LOEC	0.0867
EyC10	0.0205 (0.0141 – 0.0275)
EyC20	0.0293 (0.0208 – 0.0419)
EyC50	8.05 (0.120 – 16.4)

 

EC₅₀-Values of the Reference Item based on nominal concentrations[mg/L], (0-72 hours)

	with Headspace	without Headspace	Valid Range (average ± 3 x SD)
	Growth Rate Inhibition
ErC50	0.799	0.244	0.744±0.626
95 % confidence interval	0.707 - 1.30	0.175 - 0.350*
	Yield Inhibition
EyC50	0.607	0.236	0.409±0.208
95 % confidence interval	0.404 - 0.694	0.175 - 0.350*

SD = Standard deviation

*   = The highest concentration level without statistical significance and the lowest concentration level with statisical significance were used as confidence limits.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour algal EC50 (growth rate) was 33.1 mg/l and the EC50 (yield) was 8.05 mg/l.

Executive summary:

The acute toxicity of benzaldehyde to Pseudokirchneriella subcapitata was studied in a 72-hour toxicity study according to OECD 201. The 72-hour algal EC50 (growth rate) was 33.1 mg/l and the EC50 (yield) was 8.05 mg/l.

Description of key information

The toxicity of benzaldehyde to Pseudokirchneriella subcapitata was studied in a 72-hour toxicity study according to OECD 201. The 72-hour algal EC50 (growth rate) was 33.1 mg/l and the EC50 (yield) was 8.05 mg/l (mean measured concentration). An EC10 (growth) of 0.0389 mg/l and EC10 (yield) of 0.0205 mg/l were also reported.

Supporting studies showed 8-day NOEC (TGK) values of 20 mg/L (Bringmann and Kuhn, 1976) and 34 mg/l (Bringmann and Kuhn 1977; Bringmann and Kuhn, 1980). The outcome of a QSAR (ECOSAR) predicted an EC50 of 23 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.05 mg/L

EC10 or NOEC for freshwater algae:

0.021 mg/L