4-hydroxybenzaldehyde

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:

2017-10-05 to 2018-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations:
1.0, 3.2, 10.0, 31.6 and 100.0 mg/L

Sampling method:
Two replicates of each sample were deep-frozen (< -20 °C) directly after preparation and after 24h, 48h and 72h and stored in a freezer until analysis. Before freezing the samples after 24h, 48h and 72h exposure, the algae were separated from the medium by centrifugation at a low g-force (4000g for 20 minutes), sufficient to settle the algae. Additionally, analysis of all test item concentrations not inoculated with algae were performed. Analysis of the test item concentration at the beginning and at the end of each exposure period were performed in an analytical laboratory at the test facility using a HPLC method with UV-detection.

Vehicle:

yes

Remarks:

Reconstituted water

Details on test solutions:

PREPARATION OF THE TEST MATERIAL
A preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 1 hour. This solution was used for the preparation of the different test item concentrations. The pH value was not adjusted.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green alga
- Strain: 61.81 SAG
- Source: „Pflanzenphysiologisches Institut der Universität Göttingen", Germany.
- Method of cultivation: For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers. The cultures were shaken continuously at about 120 rpm and lit between 4440 and 8880 Lux.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

23.1 - 24.7 °C

pH:

7.03 - 8.33

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10.0, 31.6 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, fill volume: glass, 300 mL filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 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 mean light intensities were 7510 Lux ± 3.4 % and 7585 Lux ± 3.7 % prior to and at the end of the exposure period, respectively.
The mean light intensities for the samples used for analytical determination after 24 and 48 hours were 7604 Lux ± 2.7 % and 7869 Lux ± 2.9 % prior to and at the end of the exposure period, respectively.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: appr. 3.16
- Test concentrations: 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.12 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

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

12.64 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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37.93 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

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.29 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

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.85 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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.19 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, 2017).

The test item (Potassium dichromate) showed 72h EC50 of 0.91 mg/L for growth rate and 0.48 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means.

Table 1: Nominal and corresponding analytical concentrations of samples inoculated with algae

Nominal test item concentration [mg/L]	Analytical results
	0-h^1)		24-h		48-h		72-h
	Meas. Cone. [mg/L]	[%]^2)	Meas. Cone. [mg/L]	[%]^2)	Meas. Cone. [mg/L]	[%]^2)	Meas. Cone. [mg/L]	[%]^2)
0.0	0.015^3)	-	0.014^3'	-	0.014^3)	-	0.017^3)	-
1.0	0.957	95.7	0.015	1.5	0.016	1.6	0.016	1.6
3.2	3.018	94.3	1.720	53.8	0.055	1.7	0.036	1.1
10.0	9.424	94.2	8.462	84.6	0.000	<LOD	0.000	<LOD
31.6	29.741	94.1	29.210	92.4	0.000	<LOD	0.000	<LOD
100.0	93.143	93.1	92.812	92.8	94.342	94.3	93.081	93.1

LOD = Limit of detection (0.002 mg/L)

¹⁾ Measured without algae

²⁾ Rounded to the first decimal place

³⁾ Due to ingredient(s) of the algae medium, not related to test item

 

Table 2: Nominal and corresponding concentrations of analytical control samples without algae

Nominal test item concentration [mg/L]	Analytical results
	0-h		24-h		48-h		72-h
	Meas. Cone. [mg/L]	[%]^1)	Meas. Cone. [mg/L]	[%]^1)	Meas. Cone. [mg/L]	[%]^1)	Meas. Cone. [mg/L]	[%]^1)
1.0	0.957	95.7	0.928	92.8	0.933	93.3	0.906	90.6
3.2	3.018	94.3	2.997	93.7	3.031	94.7	3.001	93.8
10.0	9.424	94.2	9.453	94.5	9.587	95.9	9.475	94.8
31.6	29.741	94.1	30.171	95.5	31.979	101.2	31.530	99.8
100.0	93.143	93.1	97.832	97.8	93.897	93.9	102.346	102.3

¹⁾ Rounded to the first decimal place

 

Table 3: EC values: For the test item, the following EC values were determined:

Parameter (0-72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	7.12 (5.00-9.26)	3.29 (1.68-4.65)
EC20 [mg/L] 95% confidence interval	12.64 (9.80-15.38)	4.85 (3.00-6.35)
EC50 [mg/L] 95% confidence interval	37.93 (32.96 - 43.86)	10.19 (8.12-12.82)

 

Table 4: Algal Growth Measurement: Algal biomass (cell number) after 24, 48 and 72 hour exposure

Nominal concentration [mg/L]		Biomass of algae (Cell number per mL*)
0.0		0 hours	24 hours	48 hours	72 hours
		1.0000	4.8690	30.3480	246.3390
		1.0000	4.8267	33.3027	280.4130
		1.0000	4.9383	33.0687	269.4330
		1.0000	4.2363	27.6750	211.9230
		1.0000	4.7610	33.6960	286.6140
		1.0000	4.8177	32.4504	265.0320
	Mean	1.0000	4.7415	31.7568	259.9590
	SD	0.00000	0.25442	2.32372	27.33975
	CV	0.0	5.4	7.3	10.5
1.0		0 hours	24 hours	48 hours	72 hours
		1.0000	4.3317	32.7870	284.1930
		1.0000	4.1724	32.5737	304.6680
		1.0000	4.4397	34.7814	312.4080
	Mean	1.0000	4.3146	33.3807	300.4230
	SD	0.00000	0.13447	1.21772	14.57863
	CV	0.0	3.1	3.6	4.9
3.2		0 hours	24 hours	48 hours	72 hours
		1.0000	3.3300	21.6936	187.5420
		1.0000	3.7557	25.6086	244.2690
		1.0000	3.6360	26.7813	243.2250
	Mean	1.0000	3.5739	24.6945	225.0120
	SD	0.00000	0.21954	2.66418	32.45417
	CV	0.0	6.1	10.8	14.4
10.0		0 hours	24 hours	48 hours	72 hours
		1.0000	3.4947	14.7537	138.3867
		1.0000	3.3660	12.5217	97.4187
		1.0000	3.6387	17.2710	188.4717
	Mean	1.0000	3.4998	14.8488	141.4257
	SD	0.00000	0.13642	2.37608	45.60251
	CV	0.0	3.9	16.0	32.2
31.6		0 hours	24 hours	48 hours	72 hours
		1.0000	2.0070	10.0980	18.3600
		1.0000	1.7343	10.4904	14.4873
		1.0000	1.7370	10.9494	17.8713
	Mean	1.0000	1.8261	10.5126	16.9062
	SD	0.00000	0.15667	0.42613	2.10903
	CV	0.0	8.6	4.1	12.5
100.0		0 hours	24 hours	48 hours	72 hours
		1.0000	1.9377	2.5857	5.9904
		1.0000	1.5903	2.1960	3.6567
		1.0000	1.5507	2.1807	3.4974
	Mean	1.0000	1.6929	2.3208	4.3815
	SD	0.00000	0.21293	0.22954	1.39562
	CV	0.0	12.6	9.9	31.9

*Cell number divided by factor 10000

 

Table 5: Yield: Yield after 24, 48 and 72 hour exposure:

Nominal concentration [mg/L]	Yield
0.0		24 hours	48 hours	72 hours
		3.8690	29.3480	245.3390
		3.8267	32.3027	279.4130
		3.9383	32.0687	268.4330
		3.2363	26.6750	210.9230
		3.7610	32.6960	285.6140
		3.8177	31.4504	264.0320
	Mean	3.7415	30.7568	258.9590
	SD	0.25442	2.32372	27.33975
	CV	6.8	7.6	10.6
1.0		24 hours	48 hours	72 hours
		3.3317	31.7870	283.1930
		3.1724	31.5737	303.6680
		3.4397	33.7814	311.4080
	Mean	3.3146	32.3807	299.4230
	SD	0.13447	1.21772	14.57863
	CV	4.1	3.8	4.9
3.2		24 hours	48 hours	72 hours
		2.3300	20.6936	186.5420
		2.7557	24.6086	243.2690
		2.6360	25.7813	242.2250
	Mean	2.5739	23.6945	224.0120
	SD	0.21954	2.66418	32.45417
	CV	8.5	11.2	14.5
10.0		24 hours	48 hours	72 hours
		2.4947	13.7537	137.3867
		2.3660	11.5217	96.4187
		2.6387	16.2710	187.4717
	Mean	2.4998	13.8488	140.4257
	SD	0.13642	2.37608	45.60251
	CV	5.5	17.2	32.5
31.6		24 hours	48 hours	72 hours
		1.0070	9.0980	17.3600
		0.7343	9.4904	13.4873
		0.7370	9.9494	16.8713
	Mean	0.8261	9.5126	15.9062
	SD	0.15667	0.42613	2.10903
	CV	19.0	4.5	13.3
100.0		24 hours	48 hours	72 hours
		0.9377	1.5857	4.9904
		0.5903	1.1960	2.6567
		0.5507	1.1807	2.4974
	Mean	0.6929	1.3208	3.3815
	SD	0.21293	0.22954	1.39562
	CV	30.7	17.4	41.3

 

Table 6: Mean Specific Growth Rate - Mean specific growth rate after 24, 48 and 72 hour exposure

Nominal concentration [mg/L]	Mean specific growth rate
0.0		24 hours	48 hours	72 hours
		1.583	1.706	1.836
		1.574	1.753	1.879
		1.597	1.749	1.865
		1.444	1.660	1.785
		1.560	1.759	1.886
		1.572	1.740	1.860
	Mean	1.555	1.728	1.852
	SD	0.0559	0.0380	0.0369
	CV	3.6	2.2	2.0
1.0		24 hours	48 hours	72 hours
		1.466	1.745	1.883
		1.428	1.742	1.906
		1.491	1.775	1.915
	Mean	1.462	1.754	1.901
	SD	0.0313	0.0181	0.0163
	CV	2.1	1.0	0.9
3.2		24 hours	48 hours	72 hours
		1.203	1.539	1.745
		1.323	1.621	1.833
		1.291	1.644	1.831
	Mean	1.272	1.601	1.803
	SD	0.0622	0.0555	0.0505
	CV	4.9	3.5	2.8
		24 hours	48 hours	72 hours
		1.251	1.346	1.643
		1.214	1.264	1.526
10.0		1.292	1.425	1.746
	Mean	1.252	1.345	1.639
	SD	0.0390	0.0804	0.1101
	CV	3.1	6.0	6.7
		24 hours	48 hours	72 hours
		0.697	1.156	0.970
		0.551	1.175	0.891
31.6		0.552	1.197	0.961
	Mean	0.600	1.176	0.941
	SD	0.0839	0.0202	0.0432
	CV	14.0	1.7	4.6
		24 hours	48 hours	72 hours
		0.662	0.475	0.597
		0.464	0.393	0.432
100.0		0.439	0.390	0.417
	Mean	0.521	0.419	0.482
	SD	0.1220	0.0482	0.0996
	CV	23.4	11.5	20.7

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 72h EC50 of 37.93 mg/L for the growth rate and 10.19 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata. The 72 -h EC10 value was determined to be 7.12 mg/L for growth rate.

Executive summary:

The toxicity of the test item on the growth and growth rate of the green algae of the unicellular green alga, Pseudokirchneriella subcapitata during exposure period of 72 hours was estimated in a method according to OECD 201 under GLP. For this purpose, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to different nominal test item concentrations of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L for a period of 72 hours. The initial algae cell density in each replicate was 10 000 cells per mL. Growth of the algae was determined after 24, 48 and 72 hours exposure and compared with the growth of the control group. From the results, EC₅₀ values for growth rate and yield were calculated. Samples of the test and control media were taken directly after preparation and then daily and stored at < -20°C until further analysis of the test item concentration via HPLC with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The quantification of the test item was performed using HPLC method with UV detection. The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266. Therefore, potassium dichromate was tested as positive control (Gado, 2017).

The recoveries of the test item concentrations with algae decreased in the 1.0, 3.2, 10.0 and 31.6 mg/L test media samples over the time period from approx. 95% down to < LOD of the nominal concentrations. The recoveries of the test item concentrations in the 100.0 mg/L test media sample with algae remained stable over the testing period and are between 92.8% - 94.3%. Trace amounts of a substance were detected in the undiluted control medium at all-time points. However, this peak is most likely to have its origin in a component of the undiluted control medium and is not a contaminant of the control medium by the test item. The recoveries of the test item concentrations in all test media without algae ranged from 90.6% to 102.3% from the nominal concentrations over the time period of 72h and, thus, the test item was stable in the test medium during the study. It can be concluded from the analytical results, that the test item sticks on the algae surface, resulting in a decrease of the recoveries in the samples with algae especially at lower concentrations. Therefore, it is reasonable to conclude, that the test item is chemically stable in the test medium and that the algae are sufficiently 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.

In the control group, the biomass increased by a factor of 260.0 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 15.0%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 hours was 2.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Under the conditions of the present study, the test item showed a 72h EC50 of 37.93 mg/L for the growth rate and 10.19 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata. The 72 -h EC10 value was determined to be 7.12 mg/L for growth rate.

Description of key information

Under the conditions of the present study, the test item showed a 72h EC₅₀ of 37.93 mg/L for the growth rate and 10.19 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata. The 72h EC₁₀ value was determined to be 7.12 mg/L for growth rate (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

37.93 mg/L

EC10 or NOEC for freshwater algae:

7.12 mg/L

Additional information

The toxicity of the test item on the growth and growth rate of the green algae of the unicellular green alga, Pseudokirchneriella subcapitata during exposure period of 72 hours was estimated in a method according to OECD 201 under GLP. For this purpose, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to different nominal test item concentrations of 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L for a period of 72 hours. The initial algae cell density in each replicate was 10 000 cells per mL. Growth of the algae was determined after 24, 48 and 72 hours exposure and compared with the growth of the control group. From the results, EC₅₀ values for growth rate and yield were calculated. Samples of the test and control media were taken directly after preparation and then daily and stored at < -20 °C until further analysis of the test item concentration via HPLC with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The quantification of the test item was performed using HPLC method with UV detection. The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266. Therefore, potassium dichromate was tested as positive control (Gado, 2017).

The recoveries of the test item concentrations with algae decreased in the 1.0, 3.2, 10.0 and 31.6 mg/L test media samples over the time period from approx. 95 % down to <LOD of the nominal concentrations. The recoveries of the test item concentrations in the 100.0 mg/L test media sample with algae remained stable over the testing period and are between 92.8 % - 94.3 %. Trace amounts of a substance were detected in the undiluted control medium at all-time points. However, this peak is most likely to have its origin in a component of the undiluted control medium and is not a contaminant of the control medium by the test item. The recoveries of the test item concentrations in all test media without algae ranged from 90.6 % to 102.3 % from the nominal concentrations over the time period of 72h and, thus, the test item was stable in the test medium during the study. It can be concluded from the analytical results, that the test item sticks on the algae surface, resulting in a decrease of the recoveries in the samples with algae especially at lower concentrations. Therefore, it is reasonable to conclude, that the test item is chemically stable in the test medium and that the algae are sufficiently 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.

In the control group, the biomass increased by a factor of 260.0 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 15.0 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 hours was 2.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Under the conditions of the present study, the test item showed a 72h EC₅₀ of 37.93 mg/L for the growth rate and 10.19 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata. The 72h EC₁₀ value was determined to be 7.12 mg/L for growth rate (reference 6.1.5-1).