DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

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:

27 Aug 2018 to 13 Dec 2018

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))

Deviations:

yes

Remarks:

In the final test the contact time was 3 hours and 15 minutes, due to a malfunctioning of the stirring device. Evaluation: The deviation of the contact time is relatively small and therefore this deviation has no impact on the outcome of the test.

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Octopamine hydrochloride Appearance: White to off white powder (determined by Charles River Den Bosch) Batch: D151-1710037 Test item storage: At room temperature protected from light Stable under storage conditions until: 26 October 2019 (retest date)

Analytical monitoring:

not required

Vehicle:

yes

Remarks:

Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

Details on test solutions:

Test Item – Preparation and Test Concentrations
The batch of Octopamine hydrochloride tested was a white to off white powder with a purity of 99.8%. No correction was made for the purity/composition of the test item. A stock solution of 10 g/L was prepared by adding 5.0059 g test item to 500 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) for the combined limit range-finding test and 10.0036 g test item to 1000 mL of Milli-RO water for the final test. Magnetic stirring for 15 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 5.3 and 5.0 for the combined limit/range-finding and final test, respectively) corresponding to the test concentrations were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli - RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Any residual volumes were discarded.

Reference Item – Preparation and Test Concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol. A 1.0 g/L stock solution was prepared by dissolving 250.3 mg 3,5-dichlorophenol in Milli - RO water and making up to a total volume of 250 mL. The pH as used for the test was 8.0 in both the combined limit/range-finding and final test. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15℃) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium, resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The amount of suspended solids was determined (3.0 g/L of sludge, as used for the test). The pH was 7.4 on the day of testing. The batch of sludge was used one day after collection. Therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use. Medium Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot, The Netherlands) with the following composition: CaCl2.2H2O 211.5 mg/L MgSO4.7H2O 88.8 mg/L NaHCO3 46.7 mg/L KCl 4.2 mg/L Rationale Recognized by international guidelines as the recommended test system.

Test type:

not specified

Water media type:

not specified

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 and 22°C

pH:

The pH of the sludge was 7.2 on the day of testing.

Nominal and measured concentrations:

The test started with the preparation of a 10 g/L stock solution. Five concentrations were tested, ranging from 100 to 1000 mg/L and increasing with a factor 1.8.

Details on test conditions:

Contact time: 3 hours and 15 minutes, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium: 16 g peptone (=sewage feed) 11 g meat extract 3 g urea 0.7 g NaCl 0.4 g CaCl2.2H2O 0.2 g MgSO4.7H2O 2.8 g K2HPO4 Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
Air supply: Clean, oil-free air. Aeration The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
Performance of the test: The synthetic medium (=sewage feed, 16 mL) and an appropriate amount of the test item stock were mixed and made up to 250 mL with Milli-RO water. The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3 hour and 15 minutes contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessel. The temperature control vessel were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Concentrations: The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 100 to 1000 mg/L and increasing with a factor 1.8.

Combined Limit/Range-Finding Test In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested. The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested. Test procedure and conditions were similar to those applied in the final test with the following exceptions: The pH of the sludge was 7.2 on the day of testing. The exposure period was 3 hours.

Final Test – Test Concentrations Octopamine hydrochloride Five concentrations: 100, 180, 320, 560 and 1000 mg/L. Replicates 5 replicates per test group and 6 replicates for the control. Control Test medium without test item, treated in the same way as the test item solutions.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 180 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.:

ca. 235 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 425 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

Details on results:

Combined Limit/Range-Finding Test
The combined limit/range-finding test showed no inhibition of the respiration rate at 10 and 100 mg/L, and 36% inhibition at 1000 mg Octopamine hydrochloride per liter. Therefore, the expected EC50 was above the highest concentration tested (1000 mg/L). There was no oxygen uptake from abiotic processes. All test conditions and acceptability criteria prescribed by the study plan were met.

Final Test
Inhibition of the Respiration Rate
Due to a malfunctioning of the stirring device during the measurement of the respiration rate the respiration rate of the first replicate of the control and the first two reference item concentrations could not be determined. The two highest concentrations of the reference item were not reliable and therefor the four reference concentrations were repeated at the end of the test serie.
The effects observed were slightly higher as what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and ECx values. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 180 mg Octopamine hydrochloride per liter. At higher concentrations the inhibitory effect of Octopamine hydrochloride on aerobic waste water (activated sludge) bacteria increased from 15% at 320 mg/L to 48% at 1000 mg/L.

Determination of Effect Parameters
Parameter Octopamine hydrochloride Concentration (mg/L)
NOEC 180
EC10 235 (148 – 373)
EC20 425 (322 – 560) EC50 > 1000

Experimental Conditions
The pH in all test vessels, before addition of sludge, was between 7.2 and 7.5. After the 3 hour exposure period the pH was between 7.6 and 8.0. The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

Results with reference substance (positive control):

Due to a malfunctioning of the stirring device during the measurement of the respiration rate the respiration rate of the first replicate of the control and the first two reference item concentrations could not be determined. The two highest concentrations of the reference item were not reliable and therefor the four reference concentrations were repeated at the end of the test serie

Reported statistics and error estimates:

Calculations
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. Calculation of oxygen uptakes The respiration rate (R) from each vessel, in mg O2/L.h was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L. R was calculated by the BlueBox software as (V1 – V2)/Δt * 60 Where: V1=Value 1: the oxygen concentration at the start of the selected section of the linear phase (mg O2/L), V2=Value 2: the oxygen concentration at the end of the selected section of the linear phase (mg O2/L), Δt is the time interval between these two measurements. Negative R values were expressed as 0 mg O2/L.h (V1 5% of control respiration) a correction for this oxygen uptake was made.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test Octopamine hydrochloride was not toxic to waste water bacteria (activated sludge) at or below a concentration of 180 mg/L (NOEC).  
The EC10 was 235 mg/L (95% confidence interval: 148 - 373 mg/L).
The EC20 was 425 mg/L (95% confidence interval: 322 - 560 mg/L).
The EC50 was above 1000 mg/L.  

Executive summary:

The objective of the study was to evaluate Octopamine hydrochloride for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC). The influence of Octopamine hydrochloride on the respiration rate of activated sludge was investigated after a contact time of 3 hours and fifteen minutes. The study procedures described in this report were based on the OECD guideline No. 209, 2010. The batch of Octopamine hydrochloride tested was a white to off white powder with a purity of 99.8%.  No correction was made for the purity/composition of the test item.  The test started with the preparation of a 10 g/L stock solution.  Magnetic stirring for 15 minutes was applied to accelerate dissolution and to ensure homogeneity.  Volumes of the clear and colorless stock solution corresponding to the test concentration were then added to the test media.  Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour and 15 minutes exposure period.  Thereafter, oxygen consumption was recorded for approximately 10 minutes. The final test was performed based on the result of a preceding combined limit/range-finding test.  Five concentrations were tested, ranging from 100 to 1000 mg/L and increasing with a factor 1.8.  Five replicates per concentration and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 180 mg Octopamine hydrochloride per liter.  At higher concentrations the inhibitory effect of Octopamine hydrochloride on aerobic waste water (activated sludge) bacteria increased from 15% at 320 mg/L to 48% at 1000 mg/L. The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the study plan and was considered valid. Octopamine hydrochloride was not toxic to waste water (activated sludge) bacteria at or below a concentration of 180 mg/L (NOEC). The effect parameters for the inhibition of the respiration rate are presented below.

Parameter                     Octopamine hydrochloride Concentration (mg/L)

NOEC                            180

EC10                              235 (148 – 373)

EC20                             425 (322 – 560)

EC50                             > 1000

(   ) Between brackets the 95% confidence intervals are given.

Description of key information

Under the conditions of the test Octopamine hydrochloride was not toxic to waste water bacteria (activated sludge) at or below a concentration of 180 mg/L (NOEC).  The EC10 was 235 mg/L (95% confidence interval: 148 - 373 mg/L); the EC20 was 425 mg/L (95% confidence interval: 322 - 560 mg/L) and the EC50 was above 1000 mg/L.  

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L