Salicylaldehyde

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2021 - 28 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test sample was stored in a dark storage place at room temperature.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
In order to avoid inhalation and contact with the skin and eyes, chemically resistant gloves, a mask, safety glasses, and a white coat were worn when handling the test item.
Furthermore the test item was treated under yellow fluorescent lamps (which was removed emission less than 500 nm).

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

- Preparation of stock solution:
The test sample (200 mg) was accurately weighed by an electronic analytical balance and dissolved in purified water in order to obtain 2000 mg/L stock solution of the test item (100 mL). Three bottles of this stock solution of the test item were prepared.

-Preparation of test solutions:
a) Test mixture (3 replicates/exposure level)
The 2000 mg/L stock solution of the test item (0.7, 2.1, 6.3, 19, and 56 mL), the synthetic sewage (8 mL), the purified water (the volume subtracting the volume of the stock solution of test item from 117 mL) and the activated sludge (125 mL) were added to each test vessel.
b) Blank control (6 replicates)
The synthetic sewage (8 mL), the purified water (117 mL) and the activated sludge (125 mL) were added to each test vessel.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan.
Sampling date: December 15,2021
- Method of cultivation: In accordance with Japanese Industrial Standards (JIS) K 0102:2019
- Preparation of inoculum for exposure: The activated sludge (4 L) was fed with the synthetic sewage (200 mL) and cultivated for approximately 22 hours at 20 ± 2°C under aerobic conditions in order to acclimate the test conditions. The activated sludge was then used. Acclimation to the test item was not performed.
- Initial biomass concentration: 2970 mg/L (concentration of suspended solid)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2°C (measured value: 19.5°C)

pH:

The pH of all the test solutions was measured and confirmed to be within 7.5 ± 0.5 (measured value: 7.0-7.3).

Nominal and measured concentrations:

Preliminary test:
Nominal concentration: 10, 100, and 1000 mg/L

Definitive test:
Nominal concentration: 5.6, 17, 50, 150 and 450 mg/L (geometric series with a factor of 3)

Details on test conditions:

TEST SYSTEM
- Test vessel: Approximately 500 mL polyethylene vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 250 mL
- Aeration: Air pump (controllable flow rate at 0.5-1 L/min.)
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates): not applicable
- No. of vessels per abiotic control (replicates): not applicable
- Sludge concentration (weight of dry solids per volume): 125 mL of activated sludge to each test vessel
- Weight of dry solids per volume of reaction mixture per unit of time:


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The synthetic sewage (500 mL) was prepared at the same proportion as follows: the following reagents were dissolved in 1 L of purified water. Then, the pH of the prepared synthetic sewage was adjusted to 7.5 with 1 mol/L sodium hydroxide solution. The synthetic sewage was prepared the day before the test and was stored in a refrigerator until use.
Peptone: 16 g
Meat extract: 11 g
Urea: 3 g
Sodium chloride: 0.7 g
Calcium chloride dihydrate: 0.4 g
Magnesium sulfate heptahydrate: 0.2 g
Dipotassium hydrogen phosphate: 2.8 g

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of all the test solutions was measured and confirmed to be within 7.5 ± 0.5 (measured value: 7.0-7.3).
- Photoperiod:/
- Light intensity:/
- Details on termination of incubation:/

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Dissolved oxygen concentration in the test solution was measured with dissolved oxygen analyzer. After 3 hours of exposure, the measurement bottle was filled with the test solution promptly and softly with attention to avoid foaming. Afterwards, the concentration of dissolved oxygen (DO) in the test solution was measured continuously for 10 minutes while stirring. The test solutions (blank control) were measured in 3 replicates at the beginning and the end of the exposure, individually.
Instrument: Dissolved oxygen analyzer (No. DO-016) B-100BTi (lijima Electronics)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric series with a factor of 3
- Range finding study: 10, 100, and 1000 mg/L
- Test concentrations: 5.6,17, 50, 150, and 450 mg/L
- Results used to determine the conditions for the definitive study:
In the preliminary test, three concentrations of the test item at 10, 100, and 1000 mg/L were prepared, and their percentages of respiration inhibition were. -1%, 56%, and 95%, respectively. The results suggested that EC50 of the test item was approximately 100 mg/L and NOEC of the test item was approximately 10 mg/L. The test concentrations and the geometric factor in the definitive test were decided from the results of the preliminary study.

Reference substance (positive control):

yes

Remarks:

3,5 -Dichlorophenol (CAS n° 591-35-5; Purity: 100.0%; Supplier: FUJIFILM Wako Pure Chemical; Batch number: LKH3471; Test concentrations used for the positive control: 4.0, 8.0, 16 and 32 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

9.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

See Table 1 in 'Any other informations on results'
The activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage. This test was valid because all of the values in this test met the criteria (see “Overall remarks” for further details).
The EC50 and the EC10 of the test item were 52 mg/L and 9.3 mg/L, respectively, under the test conditions of this study. Because statistical differences were confirmed between the oxygen consumption rate of the minimum test concentration (5.6 mg/L) of the test mixture and that of the blank control, the NOEC of the test item was not obtained. It suggested that the NOEC of the test item was estimated less than 5.6 mg/L under the test conditions of this study.

Results with reference substance (positive control):

EC50 = 9.0 mg/L

Reported statistics and error estimates:

See Table 2 in 'Any other informations on results'
The results of statistical analyses of significant difference were shown Table 2. The NOEC of the test item was not obtained because statistical differences were confirmed between the oxygen consumption rate of the minimum test concentration (5.6 mg/L) of the test mixture and that of the blank control.

Table 1 Oxygen consumption rate and percentage inhibition of respiration after 3 hours of exposure

Test solution	Concentration (mg/L)	Oxygen consumption rate (mgO2/L/h)	Percentage inhibition of respiration (%)	Average percentage inhibition of respiration (%)
Test mixture	5.6	48.1,49.2,48.5	8, 5,7	7
	17	45.1,46.1,45.9	13,11,12	12
	50	24.5,24.6,24.4	53, 53, 53	53
	150	7.64, 8.15, 8.27	85, 84, 84	85
	450	4.23,4.80,4.74	92,91,91	91
Reference mixture	4.0	39.7	24	-
	8.0	29.0	44	-
	16	13.9	73	-
	32	7.89	85	-
Blank control	-	*At the beginning: 52.4,51.8, 52.6 At the end: 51.6, 50.6, 52.7	-	-

*average oxygen consumption rate in blank control: 52.0 mgO2/L/h

 

Table 2 Result of statistical analysis

Nominal concentration (mg/L)	Statistical analysis	Statistical procedure
5.6	**	Bartlett’s test One-way ANOVA Dunnett’s multiple comparison test
17	**
50	**
150	-
450	-

**: Significant difference (p<0.01)

- : The data in the exposure level was not used for statistical analysis because the concentration was greater than EC50.

Validity criteria fulfilled:

yes

Remarks:

see “Overall remarks” for further details.

Conclusions:

The EC50 and the EC10 of the test item were 52 mg/L and 9.3 mg/L, respectively, under the test conditions of this study. Because statistical differences were confirmed between the oxygen consumption rate of the minimum test concentration (5.6 mg/L) of the test mixture and that of the blank control, the NOEC of the test item was not obtained. It suggested that the NOEC of the test item was estimated less than 5.6 mg/L under the test conditions of this study.

Executive summary:

This study was performed to assess the effects on the test item to the activated sludge respiration inhibition according to OECD Test Guideline 209 with GLP compliance. 

The respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed was measured after a contact time of three hours. The respiration rate of the same activated sludge in the presence of various concentrations of the test item (nominal concentrations of 5.6, 17, 50, 150 and 450 mg/L) under otherwise identical conditions was also measured. The activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage. This test was valid because all of the values in this test met the criteria.

The EC50 and the EC10 of the test item were 52 mg/L and 9.3 mg/L, respectively, under the test conditions of this study. Because statistical differences were confirmed between the oxygen consumption rate of the minimum test concentration (5.6 mg/L) of the test mixture and that of the blank control, the NOEC of the test item was not obtained. It suggested that the NOEC of the test item was estimated less than 5.6 mg/L under the test conditions of this study.

Description of key information

3h-EC50 (activated sludge) =52 mg/L; OECD TG 209; Suzuki (2022)

3h-EC10 (activated sludge) =9.3 mg/L; OECD TG 209; Suzuki (2022)

Key value for chemical safety assessment

EC50 for microorganisms:

52 mg/L

EC10 or NOEC for microorganisms:

9.3 mg/L