(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Nov 2017 - 11 Dec 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 Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

EC Number:

814-350-4

Cas Number:

1352917-66-8

Molecular formula:

C22 H28 N Cl O4

IUPAC Name:

(2S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-aminium (2R)-(2-chlorophenyl)(hydroxy)acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: 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: 1.8 mL from the approximate centre of the test vessels at t=0 h and t=48 h.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

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 a 1 hour period of magnetic stirring 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.
- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: >2 weeks old
- Age at test initiation: <24 hours
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L (as CaCO3)

Test temperature:

19-21 °C

pH:

At t=0 h: 7.9-8.0
At t=48 h: 8.0-8.3

Dissolved oxygen:

At t=0 h: 8.6-8.9 mg/L
At t=48 h: 8.7-8.8 mg/L

Nominal and measured concentrations:

Nominal: 4.6, 10, 22, 46, 100 mg/L
Measured concentrations were 107-117% relative to nominal throughout the test (averaged over both the anionic and the cationic parts). Therefore, effect parameters were based on nominal test item concentrations. See Table 1 and Table 2 in 'Any other info on results' for details on measured concentrations throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, prepared with tap water purified by Reverse Osmosis.
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 hours and at 48 hours

RANGE-FINDING STUDY
- Test concentrations: Control, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes; the expected EC50 was between 10 and 100 mg/L (nominal concentration).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Sep 2017)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 44-73 mg/L

Details on results:

- Behavioural abnormalities: none observed in the Control group.
- Mortality of control: none
- No immobility was observed in the control or the three lowest test concentrations during the test period. After 24 hours of exposure, no immobility was observed at concentrations of 46 and 100 mg/L. At the end of the test, 35 and 100% of exposed daphnids were immobile at concentrations of 46 and 100 mg/L, respectively. See Table 3 in 'Any other information on results' for full details on immobility
- Undissolved material was observed in some of the test vessels during the test. In view of the analytical results this was not considered to be test item related.
- 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 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
- The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.
- The responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Reported statistics and error estimates:

The 48h-EC50 was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding nominal test item concentrations using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1: Final Test: Test Samples - Anion

Time of sampling [hours]	Test item concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	4.6	5.29	115
	10	12 (a)	121
	22	24.1	109
	46	51.2	111
	100	116 (a)	116
48	0	n.d.	n.a.	n.a.
	4.6	5.43	118	103
	10	11 (a)	112	93
	22	24.9	113	104
	46	53.6	116	105
	100	107	107	92

Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(a) Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a. Not applicable.

Table 2: Final Test: Test Samples - Cation

Time of sampling [hours]	Test item concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	4.6	5.23	114
	10	11 (a)	113
	22	23.7	108
	46	49.2	107
	100	114 (a)	114
48	0	n.d.	n.a.	n.a.
	4.6	5.15	112	98
	10	11 (a)	112	99
	22	23.3	106	98
	46	49.1	107	100
	100	107	107	94

Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(a) Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a. Not applicable.

Table 3: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	Nominal conc. (mg/L)
		Control	4.6	10	22	46	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0^#	0^#	0^#	0^#	0^#
	B	0	0^#	0^#	0^#	0^#	0^#
	C	0	0^#	0^#	0^#	0^#	0^#
	D	0	0^#	0^#	0^#	0^#	0^#
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0^#	0	0	2^#	5^#
	B	0	0^#	0	0^#	1^#	5^#
	C	0	0^#	0^#	0^#	2^#	5^#
	D	0	0^#	0	0^#	2^#	5
	Total immobilised	0	0	0	0	7	20
	Effect %	0	0	0	0	35	100

^#Undissolved white particles observed on the surface of the test solution.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test item was 51 mg/L, based on nominal concentrations (95% confidence interval between 44 and 73 mg/L).

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). Measured concentrations were at 107 -117 % relative to nominal throughout the test (averaged for both the anionic and cationic parts of the test substance). Therefore, effect parameters were based on nominal concentrations. No immobility was observed in the control and at concentrations of 4.6, 10 and 22 mg/L throughout the test. At the end of the test, 35 and 100% of the exposed daphnids were immobile at 46 and 100 mg/L, respectively. The 48 -hEC50 for immobility was 51 mg/L (95% confidence interval 44 -73 mg/L). The study is considered to be reliable without restrictions.