5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium chloride

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct 2017 - 29 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

other: study report amendment

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006 (Annex 5 corrected 28 July 2011)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2R)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride

Cas Number:

93601-85-5

Molecular formula:

C14H21NO.HCl

IUPAC Name:

(2R)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride

Constituent 2

Reference substance name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride (1:1)

EC Number:

695-797-0

Cas Number:

93601-86-6

Molecular formula:

C14H21NO.HCl

IUPAC Name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine hydrochloride (1:1)

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White to off-white powder
- Storage condition of test material: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test concentrations.
- Sampling method: 3.0 mL, at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: 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 an intermediate item concentration 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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to the test medium. Test solutions were prepared at the highest concentration of 100 mg/L applying approximately 1 hour of magnetic stirring, followed by 1 minute treatment with ultrasonic waves, to accelerate dissolution of the test item in 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 10^4 cells/mL.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: 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.
- 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505).
- Pre-culture medium and test medium: M2, according to OECD 201.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21.2 - 23.6°C

pH:

At t=0 h: 8.0
At t=72 h: 8.1-8.3

Nominal and measured concentrations:

Nominal: 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Measured concentrations were 103-109% of nominal throughout the test. Therefore, effect parameters were based on nominal concentrations. See Table 1 in 'Any other information on results incl. tables' for details on measured concentrations throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density (mean): 281 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 or 2 extra replicates of each test group for sampling purposes
- 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium, formulated using Milli-RO water (tap-water purified by reverse osmosis).
- Intervals of water quality measurement: pH: at the beginning and at the end of the test. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 91 to 93 μE/m2/s

EFFECT PARAMETERS MEASURED
- Cell density was measured at t= 24, 48, and 72 h.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Combined Limit/Range-Finding Test concentrations: Control, 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected ErC50 was between 1 and 10 mg/L (nominal concentration).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Sep 2017)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval 5.8-7.9 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 4.4-5.1 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on statistical significance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on biological significance

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 4.6 mg/L when compared to the control.
- Growth rates were in the range of the controls at the concentrations of 0.46 and 1.0 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to concentrations of 2.2 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at test concentrations of 2.2 mg/L and higher. However, effects were biologically relevant (i.e. >10%) only at the highest test concentration.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 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.

Reported statistics and error estimates:

Statistical significance: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
ECx: Weibull analysis using linear max. likelihood regression. The two lowest test concentrations were excluded from the analysis in order to have a better fit of the curve, since the test item showed a steep dose-response relationship at the two highest test concentrations.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	0.46	0.488	106
	1.0	1.07	107
	2.2	2.33	106
	2.2^(a)	2.37	108
	4.6	4.75	103
	10	10.4	104
24	0	n.d.	n.a.	n.a.
	0.46	0.490	106	100
	1.0	1.06	106	99
	2.2	2.34	106	100
	2.2^(a)	2.39	109	101
	4.6	4.84	105	102
	10	10.5	105	101
72	0	n.d.	n.a.	n.a.
	0.46	0.490	107	101
	1.0	1.08	108	101
	2.2	2.33	106	100
	2.2^(a)	2.40	109	101
	4.6	4.85	105	102
	10	10.7	107	102

Samples were stored in the freezer (≤ -15°C) until the day of analysis. Samples were pretreated on 28 Nov 2017 and analyzed on 29 Nov 2017. The samples were stored overnight in the autosampler at room temperature.

^(a) Without algae.

n.d. Not detected.

n.a. Not applicable.

Table 2: Growth Rate And Percentage Inhibition For The Total Test Period

Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.879	0.0205	6
0.46	1.866	0.0135	3	0.7
1.0	1.872	0.0600	3	0.4
2.2	1.794	0.0171	3	4.5^#
4.6	1.723	0.0366	3	8.3^#
10	0.000	0.0000	3	100.0*

* - effect was statistically significant

^#- effect was statistically significant but biologically not relevant (<10%)

 

Table 3: Growth Rate And Percentage Inhibition At Different Time Intervals

Nominal conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.187		1.790		1.659
0.46	3	2.176	0.5	1.826	-2.0	1.595	3.8
1.0	3	2.150	1.7	1.875	-4.7	1.590	4.1
2.2	3	2.137	2.3	1.682	6.0	1.561	5.9
4.6	3	1.977	9.6	1.675	6.4	1.517	8.6
10	3	0.238	89.1	-0.238	113.3	0.000	100.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The 72-h ErC50, ErC10 and NOErC for Pseudokirchneriella subcapitata was respectively 6.8 mg/L, 4.8 mg/L and 4.6 mg/L (based on biological relevance), based on nominal concentrations.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test substance at nominal concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg/L (triplicate) and an untreated control (6 replicates). Measured concentrations in the test solution were at 108 -117% relative to nominal throughout the test. Therefore, effect parameters were based on nominal concentrations. The EC50 for growth rate inhibition (72h-ErC50) was 6.8 mg/L, the 72h-ErC10 was 4.8 mg/L. The 72h-NOErC was 1 and 4.6 mg/L based on statistical significance and biological relevance, respectively. The study met all validity criteria, and is considered reliable without restriction.