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

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

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)

Deviations:

yes

Remarks:

The experimental start date was 10 Aug 2018. Evaluation: The experimental phase could start sooner than originally anticipated. This has no effect on the test results or study interpretation.

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:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h. Volume 1.5 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 100 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. Additionally, reserve samples of 1.5 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Details on test solutions:

The batch of Octopamine hydrochloride tested was a white to off-white powder with a purity of 99.8% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L applying a 14- minute period of magnetic stirring to accelerate dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL. Any residual volumes were discarded.

Test organisms (species):

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

Details on test organisms:

Test System
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata),
strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L K2HPO4 39.5 mg/L MgSO4.7H2O 75 mg/L Na2CO3 20 mg/L C6H8O7.H2O 6 mg/L NH4NO3 330 mg/L CaCl2.2H2O 35 mg/L C6H5FeO7.xH2O 6 mg/L H3BO3 2.9 mg/L MnCl2.4H2O 1.81 mg/L ZnCl2 0.11 mg/L CuSO4.5H2O 0.08 mg/L (NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:

NH4Cl 15 mg/L MgCl2.6H2O 12 mg/L CaCl2.2H2O 18 mg/L MgSO4.7H2O 15 mg/L KH2PO4 1.6 mg/L FeCl3.6H2O 64 µg/L Na2EDTA.2H2O 100 µg/L H3BO3 185 µg/L MnCl2.4H2O 415 µg/L ZnCl2 3 µg/L CoCl2.6H2O 1.5 µg/L CuCl2.2H2O 0.01 µg/L Na2MoO4.2H2O 7 µg/L NaHCO3 50 mg/L Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L) pH 8.1 ± 0.2

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 and 23°C

pH:

The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 units).

Nominal and measured concentrations:

Test Concentrations
Octopamine hydrochloride 0.10, 1.0, 10 and 100 mg/L.

Samples taken from the highest test concentration and the control were analysed. The concentration measured in the sample with algae was 101 mg/L at the start of the test, which decreased to 96 and 75% of the initial concentration after 24 and 72 hours of exposure, respectively. The concentration measured in the sample without algae was comparable during the first 24 hours of exposure, i.e. was 99 and 104 mg/L at the start of the test and at 24 hours of exposure, respectively. At the end of the test, the concentration had decreased more strongly than in the sample with algae and was at 10% relative to the initial concentration, indicating that the presence of algae may have a stabilizing effect on the stability of the test item.

A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures per group were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition, three replicates per group were exposed to nominally 0.10, 1.0 and 10 mg/L in the combined range-finding test

Details on test conditions:

Test System
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata),
strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L K2HPO4 39.5 mg/L MgSO4.7H2O 75 mg/L Na2CO3 20 mg/L C6H8O7.H2O 6 mg/L NH4NO3 330 mg/L CaCl2.2H2O 35 mg/L C6H5FeO7.xH2O 6 mg/L H3BO3 2.9 mg/L MnCl2.4H2O 1.81 mg/L ZnCl2 0.11 mg/L CuSO4.5H2O 0.08 mg/L (NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:

NH4Cl 15 mg/L MgCl2.6H2O 12 mg/L CaCl2.2H2O 18 mg/L MgSO4.7H2O 15 mg/L KH2PO4 1.6 mg/L FeCl3.6H2O 64 µg/L Na2EDTA.2H2O 100 µg/L H3BO3 185 µg/L MnCl2.4H2O 415 µg/L ZnCl2 3 µg/L CoCl2.6H2O 1.5 µg/L CuCl2.2H2O 0.01 µg/L Na2MoO4.2H2O 7 µg/L NaHCO3 50 mg/L Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L) pH 8.1 ± 0.2

Test Concentrations
Octopamine hydrochloride 0.10, 1.0, 10 and 100 mg/L. Control Test medium without test item or other additives. Replicates 6 replicates each of the control and the highest test group, 3 replicates of each intermediate test concentration, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration: 72 hours
Test type: Static Test vessels 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
Test Medium M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 83 to 87 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings
pH: At the beginning and at the end of the test, for the highest test concentration and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the test, microscopic observations were performed on the 100 mg/L test concentration and the control to observe for any abnormal appearance of the algae.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 90 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 90 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Details on results:

Measured Test Item Concentrations
Samples taken from the highest test concentration and the control were analysed. The concentration measured in the sample with algae was 101 mg/L at the start of the test, which decreased to 96 and 75% of the initial concentration after 24 and 72 hours of exposure, respectively. The concentration measured in the sample without algae was comparable during the first 24 hours of exposure, i.e. was 99 and 104 mg/L at the start of the test and at 24 hours of exposure, respectively. At the end of the test, the concentration had decreased more strongly than in the sample with algae and was at 10% relative to the initial concentration, indicating that the presence of algae may have a stabilizing effect on the stability of the test item. It should be noted that a small response was measured in the control at the end of the test. Considering that no concentrations could be measured at the start of the test and after 24 hours of exposure, it is assumed that the sample was contaminated during sampling or sample preparation and thus had no impact on the test results. Based on these results, the Time Weighted Average (TWA) exposure concentration in the highest test concentration was calculated to be 90 mg/L and used to express effect parameters.

Inhibition of Growth Rate and Inhibition of Yield
Inhibition of growth rates increased with increasing concentration of Octopamine hydrochloride from nominally 1.0 mg/L upwards resulting in 7.6% inhibition at the highest test concentration. The inhibition at the highest test concentration was statistically significant but below 10% and thus considered to be biologically not relevant. Inhibition of yield increased with increasing concentration of Octopamine hydrochloride from nominally 1.0 mg/L upwards resulting in 32% inhibition at the highest test concentration. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Determination of Effect Concentrations

Parameter (mg/L) NOEC* NOEC# EC10 EC20 EC50
Growth rate Value <90 90 >90 >90 >90
Yield Value <90 <90 <90 <90 >90
cl: confidence limit; * based on statistical significance; # based on biological relevance.

Experimental Conditions
During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within ±1°C).

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate and yield of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range. The EC50 for yield inhibition (72h-EYC50) was 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.

Reported statistics and error estimates:

Determination of the NOEC and Calculation of the EC50
For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for a test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (STUDENT-t test for Homogeneous Variances, α=0.05, one-sided, smaller). The ECx-values could not be determined because the observed effects were below 10%. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Octopamine hydrochloride tested. The 72h-EC10 for growth rate inhibition (ErC10) was beyond the range tested, i.e. exceeded a TWA concentration of 90 mg/L. The 72h-EC10 for yield inhibition (EyC10) was below a TWA concentration of 90 mg/L.

Executive summary:

The objective of the study was to evaluate Octopamine hydrochloride for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield. The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.  The batch of Octopamine hydrochloride tested was a white to off-white powder with a purity of 99.8% and completely soluble in test medium at the concentrations tested. A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures per group were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition, three replicates per group were exposed to nominally 0.10, 1.0 and 10 mg/L in the combined range-finding test. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.  Inhibition of growth rates and yield increased with increasing concentration of Octopamine hydrochloride from nominally 1.0 mg/L upwards resulting in 7.6 and 32% inhibition at the highest test concentration, respectively.  Samples taken from the highest test concentration and the control were analysed. The concentration measured at the start of the test was 101 mg/L and decreased to 96 and 75% of the initial concentration after 24 and 72 hours of exposure, respectively. Based on these results, the Time Weighted Average (TWA) exposure concentration in the highest test concentration was calculated to be 90 mg/L and used to express effect parameters. The study met the acceptability criteria prescribed by the study plan and was considered valid. The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)              NOEC*              NOEC#              EC10              EC20              EC50

Growth rate Value                <90                     90                     >90                  >90                 >90

Yield  Value                        <90                     <90                   <90 <90 >90

cl – confidence limit; * based on statistical significance; # based on biological relevance

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Octopamine hydrochloride tested. The 72h-EC10 for growth rate inhibition (ErC10) was beyond the range tested, i.e. exceeded a TWA concentration of 90 mg/L. The 72h-EC10 for yield inhibition (EyC10) was below a TWA concentration of 90 mg/L.

Description of key information

Under the conditions of the study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Octopamine hydrochloride tested. The 72h-EC10 for growth rate inhibition (ErC10) was beyond the range tested, i.e. exceeded a TWA concentration of 90 mg/L. The 72h-EC10 for yield inhibition (EyC10) was below a TWA concentration of 90 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

90 mg/L