2-bromo-5-hydroxy-4-methoxybenzaldehyde

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-02-19 to 1998-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC (Activated Sludge Respiration Inhibition Test), Official Journal of the European Communities No. L 133/118-122

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate issued by the Swiss GLP monitoring authorities.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

426-540-0

EC Name:

-

Cas Number:

2973-59-3

Molecular formula:

C8 H7 Br O3

IUPAC Name:

2-bromo-5-hydroxy-4-methoxybenzaldehyde

Details on test material:

- Name of test material (as cited in study report): T002019
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: solid
- Analytical purity: 91.3% by High Performance Liquid Chromatography (HPLC)
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 00265786
- Expiration date of the lot/batch: 1998-07-01
- Stability under test conditions: no data
- Storage condition of test material: at room temperature at about 20 deg C, in the dark
- Other: no data

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00265786
- Expiration date of the lot/batch: 1998-07-01 (retesting date)
- Purity : 91.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20°C, in the dark
- Stability under test conditions: was determined within RCC project No. 676023
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: 70 mg/L (was determined within RCC Project No. 675775 and found to be 123 mg/L (± 3.6 mg/L) at 20 ± 1 °C.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Not applicable

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Test substance amounts of 5, 16, 50, 160 and 500 mg were directly weighed by means of an analytical balance into 1000-mL glass flasks containing 284 mL of deionized water. The test substance was mixed with the deionized water by ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark. Immediately prior to test start (incubation), 16 mL of synthetic sewage feed and 200 mL of sludge inoculum were added, resulting in a final volume of 500 mL.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: yes, deionized water negative controls were included
- Evidence of undissolved material: no

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: wastewater treatment plant (ARA Ergolz II, Füllindorf, Switzerland) treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was washed by centrifugation, the supernatant liquid phase was decanted and the solid material resuspended in tap water. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet sludge to its dry weight determined. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to a mixed liquor of suspended solids (MLSS) of 3 g/L (on dry weight basis). During holding, the sludge was fed daily, starting two days prior to use, with 50 mL synthetic sewage/L and was kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was determined to be 2.9 g/L. The pH of the activated sludge inoculum was adjusted to 7.2 with 0.5 mol H2SO4.
- Pretreatment: no
- Initial biomass concentration: 2.9 g/L MLSS (on dry weight basis)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

none

Test conditions

Hardness:

no data

Test temperature:

21 °C (start of incubation), 19 °C (end of incubation)

pH:

pH= 8.2 at start of test.
pH= 8.7 at end of test.

Dissolved oxygen:

at least 6.0 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal: 10, 32, 100, 320 and 1000 mg test substance/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass
- Type: open
- Material, size, headspace, fill volume: glass, 1000 mL, 500 mL, 500 mL
- Aeration: All test vessels were continuously aerated with compressed air at an air-flow rate of about 1 L/min.
- Type of flow-through: not applicable
- Renewal rate of test solution: not applicable
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 2.9 g/L MLSS

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the activated sludge inoculum was adjusted to 7.2 with 0.5 mol/L H2SO4
- Photoperiod: no data
- Light intensity: no data


EFFECT PARAMETERS MEASURED:
- The respiration rate of the activated sludge inoculum was measured immediately following the 3-hour incubation period. For measurement of the respiration rate a well-mixed sample of each test flask was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated. The oxygen concentration was measured with an oxygen electrode and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the linear part of the respiration curve, or in case of a low oxygen consumption over a period of about ten minutes. The inhibitory effect of the test substance or of the reference substance on the respiration rate (oxygen consumption per minute) is expressed as a percentage of the mean respiration rate of the two controls measured at the start and at the end of the test series (flasks 1 and 10):
(1 - 2Rs/[Rc1 + Rc2]) x 100% = percent inhibition,
where Rs = oxygen consumption rate (in mg O2/L/minute) at tested concentration; Rd = oxygen consumption rate in control flask 1; and Rc2 = oxygen consumption rate in control flask 10.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: not applicable
Range finding study
- Test concentrations: not applicable
- Results used to determine the conditions for the definitive study: not applicable

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (10, 32 and 100 mg/L)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

328 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI: 145-668 mg/L

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No. From the appearance of the test substance in the final test medium, beige homogeneous suspensions were noted for all test concentrations.
- Effect concentrations exceeding solubility of substance in test medium: no, concentrations exceeding 1000 mg/L nominal were not tested
- Up to and including the concentration of nominal 100 mg/L the test substance had no significant (<15%) inhibitory effect on the respiration rate of activated sludge after an incubation period of three hours. At the next higher test substance concentrations of nominal 320 and 1000 mg/L the respiration rate was moderately inhibited by 27.2% and 38.1%, respectively. The saturation concentration (equilibrium) was reached under the present test and resulted in less than 50% inhibition after three hours contact time. Thus, the 3-hour EC50 of the test substance is clearly higher than the limit of water solubility under the present test conditions, or >1000 mg/L nominal. The 3-hour EC20 was determined by Probit analysis to be 328 mg/L with 95% confidence limits from 145 to 668 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid?: yes
- Relevant effect levels: Compared to the controls the respiration rate of the activated sludge was only slightly inhibited (17.2%) at the lowest nominal concentration of 10 mg/L. At the next higher nominal concentrations of 32 and 100 mg/L, the respiration rate was inhibited by 76.6 and 92.1%, respectively. The 3-hour EC50 of the positive control substance 3,5-dichlorophenol was calculated to be 20.9 mg/L (95% confidence limits were not calculated).
- Other: no data

Reported statistics and error estimates:

- The effect concentrations were determined by Probit analysis.

Any other information on results incl. tables

Table 1. Oxygen consumption of activated sludge
Flask No.	Chemical	Concentration of test chemical (mg/L)	Oxygen consumption (mg O2/L/min)	Inhibition (%)	pH Values		Oxygen conc. (mg O2/L)
					Start	End	Start	End
1	Control		0.625		8.2	8.7	6.2	7.7
10	Control		0.667		8.2	8.7	6.2	7.4
Mean			0.646
Deviation (%)			6.7
5	T002019	1000	0.400	38.1	8.2	8.7	6.1	8.2
6	T002019	320	0.470	27.2	8.2	8.7	6.0	8.1
7	T002019	100	0.611	5.4	8.2	8.7	6.0	7.7
8	T002019	32	0.672	-4.0	8.2	8.7	6.1	7.3
9	T002019	10	0.675	-4.5	8.2	8.7	6.1	7.2

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance was assessed for its toxicity to aerobic waste water microorganisms of activated sludge in a 3-hour respiration test (according OECD guideline 209) up to a nominal concentration of 1000 mg/L which is well above the water solubility limit. The 3-hour EC50 of the test substance could not be quantified because the saturation concentration (equilibrium) under the present test conditions was reached and resulted in less than 15% inhibition after three hours contact time. The 3-hour EC20 was calculated to be 329 mg/L based on nominal test substance concentrations. The results of the study can be considered reliable without restriction.